Caged RNAs and methods of use thereof

ABSTRACT

Methods of using labeled interfering RNAs to detect and/or quantitate target mRNAs in cells are provided. Related compositions, systems, and kits are also provided. Caged interfering RNAs (e.g., photoactivatable interfering RNAs), methods of using such caged RNAs, and related systems and kits are also provided. Caged RNAs capable of repressing translation of a target mRNA or silencing transcription of a target gene are also provided, along with related methods, systems, and kits. Methods and compositions for introducing RNAs into cells, using RNAs covalently associated with protein transduction domains and/or lipids, are provided. Also provided are methods and compositions for selectively attenuating expression of a target mRNA by controlling expression of an interfering RNA, an RNA capable of initiating translation repression, or an RNA capable of initiating transcriptional silencing.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. ProvisionalPatent Application No. 60/572,564, filed May 18, 2004, entitled“RNAi-based Sensors, Caged RNAs, and Methods of Use Thereof” by Nguyenet al., and is a continuation-in-part of U.S. patent application Ser.No. 10/716,393, filed Nov. 17, 2003, entitled “RNAi-based Sensors, CagedInterfering RNAs, and Methods of Use Thereof” by Nguyen and McMaster,which claims priority to and benefit of the following prior provisionalpatent applications: U.S. Ser. No. 60/427,664, filed Nov. 18, 2002,entitled “Photo Activated Sensors, Regulators and Compounds” by Nguyenand McMaster, U.S. Ser. No. 60/436,855, filed Dec. 26, 2002, entitled“Caged Sensors, Regulators and Compounds and Uses Thereof” by Nguyen andMcMaster, U.S. Ser. No. 60/439,917, filed Jan. 13, 2003, entitled “CagedSensors, Regulators and Compounds and Uses Thereof” by Nguyen andMcMaster, U.S. Ser. No. 60/451,177, filed Feb. 27, 2003, entitled “CagedSensors, Regulators and Compounds and Uses Thereof” by Nguyen et al.,U.S. Ser. No. 60/456,870, filed Mar. 21, 2003, entitled “Caged Sensors,Regulators and Compounds and Uses Thereof” by Nguyen et al.; U.S. Ser.No. 60/484,785, filed Jul. 3, 2003, entitled “RNAi-based Sensors andMethods of Use Thereof” by Nguyen and McMaster, and U.S. Ser. No.60/501,599 filed Sep. 9, 2003, entitled “Caged Sensors, Regulators andCompounds and Uses Thereof” by Nguyen et al., each of which isincorporated herein by reference in its entirety for all purposes.

FIELD OF THE INVENTION

This invention is in the field of caged RNA, e.g., photoactivatablecaged RNA. The invention relates to caged RNAs capable of repressingtranslation of a target mRNA and/or silencing transcription of a targetgene and use of such caged RNAs to precisely control, spatially and/ortemporally, expression of the target mRNA or gene. Methods andcompositions for selectively attenuating expression of a target mRNA bycontrolling expression of an interfering RNA or an RNA that mediatestranslational repression are also provided. The invention also relatesto introduction of single- or double-stranded RNAs into cells.

BACKGROUND OF THE INVENTION

A number of experimental designs in basic research, clinical diagnosis,drug discovery, and the like involve the detection, and frequently alsothe quantitation, of a particular mRNA. However, current methods fordetecting and/or measuring mRNA transcripts from cells (such as Northernblot, quantitative rt-PCR, microarray, branched DNA, and in situhybridization assays) generally require the cells to be lysed or fixed.In addition, most current methods require that mRNA purification and/orreverse transcription be performed. Furthermore, current methodstypically involve multi-step processing that contributes to high intra-and inter-assay variation. Consequently, current methods do not providelive, dynamic, and location-specific imaging and measurements of mRNA,because the cells are exposed to environmental changes.

Cellular assays are critical tools in the drug discovery process and inbasic research. In the future, these assays will play a major role insystems biology, permitting the examination of cell structure andfunction and the determination of a drug compound's ability to enter acell, the compound's toxicity and its overall efficacy. Advances inimaging technologies, fluorescent probes, and assay automation arepredicted to drive the worldwide cellular assays market from anestimated $300 million in 2002 to $500 million in 2007. The most commonapplication for cellular assay technology in drug discovery is targetvalidation and lead identification and optimization. However, thecomplexity and richness in cellular assay data sets, compared togenomics and proteomics studies, will provide scientists withunparalleled tools to aid discovery efforts throughout the discoveryprocess and for basic research applications.

To achieve the goal of measuring the spectrum of molecular events incells, there is definite need for “in cell sensor probes” thatquantitatively measure protein (or other) activities, mRNA levels, orthe like, directly in cells in a regulated fashion to give real timefunctional data, without using expression vectors. These “in cell sensorprobes” could be used to define pathways in a Parallel QuantitativeBiology (PQB) format for systems biology, providing novel regulatedcell-based functional screening in a high throughput mode. Such probes,termed PAC probes (PhotoActivated Cell probes), are described herein,for example, probes that comprise interfering RNAs. The invention alsoprovides other benefits which will become apparent upon review of thedisclosure. A complete understanding of the invention will be obtainedupon review of the following.

SUMMARY OF THE INVENTION

The present invention provides methods, compositions, and kits includingcaged RNAs (e.g., photoactivatable caged RNAs). For example, theinvention provides caged RNAs capable of repressing translation of atarget mRNA or silencing transcription of a target gene, as well asmethods of using such caged RNAs to selectively attenuate expression oftarget genes. In addition, the invention provides methods andcompositions for introducing RNAs into cells. Methods and compositionsfor selectively attenuating expression of a target mRNA by controllingexpression of an RNA capable of repressing translation of the targetmRNA are also provided.

One general class of embodiments provides a composition comprising acaged RNA. The caged RNA includes an RNA capable of repressingtranslation of a target mRNA. The caged RNA also includes one or morefirst caging groups associated with the RNA. The first caging groupsinhibit (e.g., prevent) the RNA from repressing translation of thetarget mRNA in a cell comprising the caged RNA. Typically, removal of oran induced conformational change in the first caging groups permits theRNA to repress translation of the target mRNA. In a preferred class ofembodiments, the RNA does not initiate degradation of the target mRNA ina cell comprising the RNA.

The RNA can have any of a variety of structures, lengths, and/or thelike. For example, the RNA can be single-stranded, or, preferably,double-stranded. The RNA typically comprises at least an antisensestrand (e.g., only the antisense strand if the RNA is single-stranded,or the antisense strand and a complementary or partially complementarysense strand if the RNA is double-stranded). In one class ofembodiments, the antisense strand of the RNA comprises a first regionwhich is complementary to a second region of the target mRNA. The firstregion is interrupted by one or more nucleotides which are notcomplementary to the second region; for example, one or more nucleotideswhich form a bulge when the antisense strand binds the target mRNA. Thefirst region is optionally interrupted by two, three, four, or morenucleotides which are not complementary to the second region.

The second region, the region of the mRNA to which the antisense strandbinds, can be located essentially anywhere within the mRNA, e.g., the 5′UTR, an exon, an exon, an exon-intron boundary, or the like. In oneclass of embodiments, the second region is within the 3′ UTR of thetarget mRNA. The target mRNA optionally includes a plurality of repeatsof the second region, e.g., tandem repeats, and/or a regioncomplementary to a different RNA capable of repressing translation ofthe target.

The RNA optionally comprises at least one double-stranded region thatincludes the antisense strand and a sense strand. The sense strand canbe completely complementary to the antisense strand over thedouble-stranded region. Alternatively, in some embodiments, the sensestrand is not completely complementary to the antisense strand over thedouble-stranded region. For example, the double-stranded region caninclude one or more mismatches, bulges, loops, and/or the like. Themismatched nucleotides can be the same or different nucleotides thanthose mismatched to the target mRNA.

In certain embodiments, the RNA comprises a first polyribonucleotidecomprising the sense strand and a second polyribonucleotide comprisingthe antisense strand. For example, the first polyribonucleotide cancomprise between 17 and 29 nucleotides, the second polyribonucleotidecan comprise between 17 and 29 nucleotides, and the double-strandedregion can comprise between 17 and 29 base pairs. The first and secondpolyribonucleotides can form a duplex over their entire length, or theycan have overhangs (e.g., 5′ or 3′ overhangs). For example, in someembodiments, the first polyribonucleotide and the secondpolyribonucleotide each comprise a two nucleotide TT 3′ overhang (whereT is 2′-deoxythymidine). In one class of embodiments, at least one ofthe one or more first caging groups is optionally covalently attached toa 5′ hydroxyl or a 5′ phosphate of the second polyribonucleotide. Thefirst caging group is optionally covalently attached to the firstpolyribonucleotide and to the second polyribonucleotide, e.g., to the 3′end of the first polyribonucleotide and to the 5′ end of the secondpolyribonucleotide. Instead of comprising two polyribonucleotides, insome embodiments, the RNA comprises a self-complementarypolyribonucleotide (e.g., a shRNA).

The composition optionally also includes the target mRNA and/or a cell.For example, the composition can include a cell comprising the targetmRNA and/or the caged RNA.

The one or more first caging groups associated with the RNA can becovalently or non-covalently attached to the RNA. In a preferred aspect,the one or more first caging groups are photoactivatable (e.g.,photolabile). Other caging groups are removable via input of differentuncaging energies; e.g., the one or more caging groups can be removableby sonication or application of heat, or can be removed by a chemical orenzyme. In some embodiments, the one or more first caging groups eachcomprises a first binding moiety, and the composition includes a secondbinding moiety that can bind at least one of the first binding moieties.

In some embodiments, the RNA also includes at least one label, e.g., afluorescent label. Optionally, binding and/or repression of translationof the target mRNA by the RNA results in a binding and/orrepression-dependent change in a signal output of the label.

In one class of embodiments, the RNA is associated with a cellulardelivery module that can mediate introduction of the RNA into a cell.The cellular delivery module can comprise, e.g., a polypeptide or alipid. The cellular delivery module is optionally covalently attached tothe RNA, e.g., through a disulfide bond or a covalent attachment whichis reversible by exposure to light of a preselected wavelength. Thecellular delivery module is optionally associated with one or moresecond caging groups which inhibit the cellular delivery module frommediating introduction of the RNA into a cell. The cellular deliverymodule optionally serves as the first caging group. In one class ofembodiments, the RNA comprises a first polyribonucleotide comprising asense strand and a second polyribonucleotide comprising an antisensestrand, and a cellular delivery module is covalently attached to thesecond polyribonucleotide.

Optionally, in the embodiments herein, the caged RNA is bound to amatrix (e.g., electrostatically, covalently, directly or via a linker).In one aspect, the matrix is a surface and the RNA is bound to thesurface at a predetermined location within an array comprising otherRNAs. In other embodiments, the matrix comprises a bead (e.g.,color-coded or otherwise addressable).

Kits for making the caged RNA are also a feature of the invention. Thus,one class of embodiments provides a kit including an RNA, one or morefirst caging groups, and instructions for assembling the RNA and thefirst caging groups to form the caged RNA, packaged in one or morecontainers. Another class of embodiments provides a kit comprising oneor more first caging groups and instructions for assembling the firstcaging groups and an RNA supplied by a user of the kit to form the cagedRNA, packaged in one or more containers.

Another general class embodiments provides methods of selectivelyattenuating expression of a target gene in a cell. In the methods, acaged RNA is introduced into the cell. The caged RNA includes an RNAcapable of repressing translation of a target mRNA transcribed from thetarget gene. The caged RNA also comprises one or more caging groupsassociated with the RNA, the caging groups inhibiting (e.g., preventing)the RNA from repressing translation of the target mRNA in the cell.Repression of translation is initiated by exposing the cell to uncagingenergy (e.g., light of a predetermined wavelength), freeing the RNA frominhibition by the caging groups. In a preferred class of embodiments,the amount of the target mRNA present in the cell is not affected by thepresence of the RNA in the cell; i.e., uncaging the RNA does notinitiate RNAi.

Exposing the cell to uncaging energy optionally includes exposing thecell to light of a first wavelength. This exposure can be addressable;e.g., the caged RNA can be exposed to light of the first wavelength byexposing one or more preselected areas (e.g., wells of a microtiterplate or portions thereof, or the like) to the light. As anotherexample, the uncaging energy can be directed at a preselected subset ofa cell population comprising the cell.

Exposing the cell to light of the first wavelength optionally comprisesexposing the cell to light such that the intensity of the light and theduration of exposure to the light are controlled such that a firstportion (which can be a selected amount) of the caged RNA is uncaged anda second portion of the caged RNA remains caged. Furthermore, theuncaging step can be repeated until the caged RNA is depleted.

Caging the RNA permits temporal control over initiation of translationalrepression. For example, the method can include contacting the cell anda test compound and exposing the cell to the uncaging energy at apreselected time point with respect to a time at which the cell and thetest compound are contacted.

Essentially all of the features noted for the compositions above applyto these methods as well, as relevant. For example, in one class ofembodiments, the caged RNA comprises a cellular delivery module that canmediate introduction of the caged RNA into the cell, the cellulardelivery module being associated with the RNA. In this class ofembodiments, the caged RNA is introduced into the cell by contacting thecell with the caged RNA associated with the cellular delivery module. Asanother example, in certain embodiments, the RNA comprises at least onelabel, and the methods include detecting a signal from the label.

Another aspect of the invention relates to RNAs capable of inducinghistone methylation and chromatin silencing. Thus, one general class ofembodiments provides a caged RNA that includes an RNA capable ofsilencing transcription of a target gene and one or more first caginggroups associated with the RNA. The first caging groups inhibit (e.g.,prevent) the RNA from silencing transcription of the target gene in acell comprising the caged RNA.

Essentially all of the various optional configurations and featuresnoted for the embodiments above apply here as well, to the extent theyare relevant, e.g., for percent inhibition by the caging groups,structure of the RNA, label configurations (e.g., use of fluorescentlabels, fluorescent label/quencher, and donor/acceptor combinations),signal output types, use of caging groups (e.g., photolabile caginggroups), appropriate uncaging energies (light, heat, sonic, etc.), useof cellular delivery modules (e.g., amphipathic peptides, cationicpeptides, protein transduction domains, and lipids), and the like. Inanother aspect, systems and/or apparatus comprising the compositions(e.g., the caged RNAs) noted above and, e.g., components such asdetectors, fluid handling apparatus, sources of uncaging energy, or thelike, are a feature of the invention, as are kits for making or usingthe caged RNAs.

Another general class of embodiments provides methods of selectivelyattenuating expression of a target gene in a cell. In the methods, acaged RNA is introduced into the cell. The caged RNA comprises an RNAcapable of silencing transcription of the target gene. The caged RNAalso includes one or more first caging groups associated with the RNAthat inhibit (e.g., prevent) the RNA from silencing transcription of thetarget gene in the cell. Silencing of transcription of the target geneis initiated by exposing the cell to uncaging energy, freeing the RNAfrom inhibition by the caging groups.

Essentially all of the features noted for the embodiments above apply tothese methods as well, as relevant, e.g., for types of uncaging energy,temporal and spatial control of uncaging, introduction of the RNA intothe cell through use of a cellular delivery module, label detection, andthe like.

In another general class of methods for selectively attenuatingexpression of a gene in a cell, a first caged DNA and a second caged DNAare introduced into the cell. The first caged DNA includes a first DNAencoding an RNA sense strand and one or more caging groups. The secondcaged DNA comprises a second DNA encoding an RNA antisense strand andone or more caging groups. The presence of the caging groups preventstranscription of the first and second DNAs, the first and second DNAseach comprising at least a portion of the target gene, and the sense andantisense strands being at least partially complementary and able toform a duplex over at least a portion of their lengths. Translationalrepression is initiated by generating double-stranded RNA by exposingthe cell to uncaging energy, whereby exposure to the uncaging energyfrees the first and second DNAs from the caging groups and permitstranscription of the first and second DNAs to occur. All of the aboveoptional method variations apply to this method as well, to the extentthey are relevant. Further, the various composition components notedabove can be adapted for use in this method, as appropriate; e.g., withrespect to configuration of the RNA, use of caging groups (e.g.,photolabile caging groups), appropriate uncaging energies (light, heat,sonic, etc.), use of cellular delivery modules (e.g., amphipathicpeptides, protein transduction domains, and lipids), and the like.

Protein transduction domains can be used to introduce RNAs, includingcaged RNAs, into cells. Thus, one class of embodiments provides acomposition comprising a protein transduction domain covalently attachedto an RNA. The RNA can comprise at least one double-stranded region, thedouble-stranded region comprising a sense strand and an antisensestrand, the antisense strand comprising a region which is substantiallycomplementary to a region of a target mRNA. Alternatively, the RNA cancomprise a single polyribonucleotide strand comprising an antisensestrand, the antisense strand comprising a region which is substantiallycomplementary to a region of a target mRNA.

The RNA can have any of a variety of structures, lengths, and/or thelike. Thus, in one class of embodiments, the region of the antisensestrand which is substantially complementary to the region of the targetmRNA is completely complementary to the region of the target mRNA. Inanother class of embodiments, the region of the antisense strand whichis substantially complementary to the region of the target mRNAcomprises at least a first and a second subregion, each of which iscompletely complementary to the target mRNA, flanking one or morenucleotides (e.g., two, three, four, or more nucleotides) which are notcomplementary to the target mRNA.

Essentially all of the features noted for the embodiments above apply tothese methods as well, as relevant. For example, one or more firstcaging groups can be associated with the RNA, inhibiting (e.g.,preventing) the RNA from repressing translation of the target mRNA in acell. The composition is optionally also includes the target mRNA and/ora cell, e.g., a cell comprising the target mRNA and/or the RNA.

The invention also provides related methods of introducing an RNA into acell. In the methods, a composition comprising an RNA and a proteintransduction domain covalently attached to the RNA is provided. The RNAcan comprise at least one double-stranded region, the double-strandedregion comprising a sense strand and an antisense strand, the antisensestrand comprising a region which is substantially complementary to aregion of a target mRNA. Alternatively, the RNA can comprise a singlepolyribonucleotide strand comprising an antisense strand, the antisensestrand comprising a region which is substantially complementary to aregion of a target mRNA. The composition and the cell are contacted,whereby the protein transduction domain mediates introduction of the RNAinto the cell.

Essentially all of the features noted for the embodiments above apply tothese methods as well, as relevant. For example, the RNA can be caged.Thus, in one class of embodiments, one or more first caging groups areassociated with the RNA, inhibiting (e.g., preventing) the RNA fromrepressing translation of the target mRNA in the cell, and the methodsinclude initiating translational repression of the target mRNA byexposing the cell to uncaging energy of a first type, whereby exposureto the uncaging energy frees the RNA from inhibition by the first caginggroups.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Panels A-I schematically illustrate example interfering RNAsensors. The top strand corresponds to the sense strand and the bottomthe antisense strand. Boxes A and B represent either a fluorescent labeland a quencher (or vice versa) or a donor and acceptor (or vice versa).

FIG. 2 schematically illustrates multiplexed interfering RNA sensorshaving different fluorescent label (F)/quencher (Q) combinations.

FIG. 3 schematically illustrates multiplexed interfering RNA sensors,one example with a donor/acceptor combination (F1/F2), one example witha donor/acceptor combination suitable for TR-FRET (APC/Eu), and oneexample with a fluorescent label/quencher (F5/Q) combination.

FIG. 4 schematically illustrates caged siRNAs with a photolabile linkerbetween the two strands. The top strand corresponds to the sense strandand the bottom the antisense strand.

FIG. 5 schematically illustrates example caged siRNAs. The top strandcorresponds to the sense strand and the bottom the antisense strand.

FIG. 6 schematically illustrates example caged siRNAs. The top strandcorresponds to the sense strand and the bottom the antisense strand.

FIG. 7 schematically illustrates example siRNAs in which a cellulardelivery module is attached to the siRNA by a photolabile linker. Thetop strand corresponds to the sense strand and the bottom the antisensestrand.

FIG. 8 schematically illustrates example siRNAs in which a proteincarrier is attached to the siRNA by a photolabile linker. The top strandcorresponds to the sense strand and the bottom the antisense strand.

FIG. 9 schematically illustrates an example siRNA in which a proteintransduction domain- and endosomal release agent-coated bead is attachedto the siRNA by a photolabile linker. The top strand corresponds to thesense strand and the bottom the antisense strand.

FIG. 10 schematically illustrates an example siRNA in which a lipid isattached to the siRNA by a photolabile linker. The top strandcorresponds to the sense strand and the bottom the antisense strand.

FIG. 11 schematically illustrates caging of an siRNA in a photolabilevesicle (solid oval) associated with a protein transduction domain(PTD). The siRNA is released when the vesicle dissociates (broken oval)after exposure to light.

FIG. 12 schematically illustrates caged siRNAs that can be used, e.g.,as probes for mRNA.

FIG. 13 schematically illustrates caged shRNAs that can be used, e.g.,as probes for mRNA.

FIG. 14 schematically depicts a caged siRNA linked with a peptidetransport moiety.

FIG. 15 schematically illustrates measurement of mRNA with a FRET siRNA.

FIG. 16 schematically illustrates the use of multiple siRNAs per targetgene.

FIG. 17 depicts a flowchart illustrating an example workflow for assaysusing photoactivatable mRNA sensors in a live cell assay format.

FIG. 18 schematically depicts the detection of splice variants withsiRNA sensors that span splice junctions.

FIG. 19 presents the sequence of a portion of human GAPDH (SEQ ID NO:1).The positions of the sense strand of three interfering RNAs againstGAPDH (RNAi 1-RNAi 3) are also indicated.

FIG. 20 Panel A schematically illustrates an annealed GAPDH interferingRNA sensor; Panel B schematically illustrates a denatured GAPDHinterfering RNA sensor; and Panel C shows fluorescent emission spectrafor the antisense strand (curve 1), the sense strand (curve 2), and theannealed strands (curve 3) of a GAPDH interfering RNA sensor.

FIG. 21 shows the GAPDH mRNA level as measured by a bDNA assay at theindicated time points after lipofection of labeled RNAi 1 (Panel A), ascompared to a negative control (Panel B, no lipofection reagent).

FIG. 22 compares the percentage knockout of GAPDH expression, asmeasured by the bDNA assay, for labeled RNAi 1-3.

FIG. 23 shows the results of bDNA assays (RLU, luminescence) compared toFITC signals (FLU) for cells lipofected with the RNAi 1 (Panel A), RNAi2 (Panel B), and RNAi 3 (Panel C) sensors.

FIG. 24 shows the ratio of the bDNA assay measurement of GAPDH mRNAlevels at 20 h/4 h and the ratio of the FITC signal from labeled RNAi's1-3 at 20 h/4 h.

FIG. 25 Panel A presents a graph of the results of bDNA assays (RLU,relative luminescent units, indicating the GAPDH mRNA level) and a graphof the fluorescent signal (RFU, relative fluorescence units) from thelabeled RNAi sensor, at the indicated time points after lipofection ofthe sensor. Panel B presents a graph of the fluorescent signals againstthe bDNA assay results.

FIG. 26 schematically illustrates use of an environmentally responsivepolymer as a noncovalently associated caging group.

FIG. 27 schematically illustrates use of an environmentally responsivepolymer as a covalently associated caging group for an siRNA. The topstrand of the RNA corresponds to the sense strand and the bottom theantisense strand.

FIG. 28 Panel A schematically illustrates the caged double-strandedsiRNA RNAi 1. Panel B depicts the caged antisense strand of caged RNAi 1(SEQ ID NO:2).

FIG. 29 Panel A presents a graph of GAPDH expression relative tocyclophilin expression in untransfected cells, cells transfected withRNAi 1, cells transfected with in vitro uncaged caged RNAi 1, cellstransfected with caged RNAi 1 but not exposed to UV light, and cellstransfected with caged RNAi 1 and exposed to UV light, as measured by abDNA assay. Panel B presents a graph of relative GAPDH expression levelsin cells transfected with RNAi 1, transfected with in vitro uncagedcaged RNAi 1, transfected with caged RNAi 1 but not exposed to UV light,and transfected with caged RNAi 1 and exposed to UV light, as measuredby a bDNA assay and normalized to the expression level in cellstransfected with RNAi 1.

FIG. 30 schematically illustrates induction of expression of aninterfering RNA by uncaging of a first activation component,tetracycline in this example.

FIG. 31 schematically illustrates induction of expression of aninterfering RNA by uncaging of a first activation component, IP3 in thisexample.

FIG. 32 schematically illustrates processing of an example pre-miRNA toform an miRNA and binding of the miRNA antisense strand to the 3′UTR ofan mRNA.

FIG. 33 presents a graph of relative GAPDH expression in cellstransfected with a scrambled negative control GAPDH siRNAi, cellstransfected with caged RNAi 1 but not exposed to UV light, cellstransfected with in vitro uncaged caged RNAi 1, and cells transfectedwith caged RNAi 1 and exposed to UV light.

FIG. 34 presents a graph of normalized GAPDH expression in cellstransfected with caged RNAi 1 and exposed to varying doses of UV light.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. The following definitionssupplement those in the art and are directed to the current applicationand are not to be imputed to any related or unrelated case, e.g., to anycommonly owned patent or application. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice for testing of the present invention, the preferred materialsand methods are described herein. Accordingly, the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to be limiting.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a protein”includes a plurality of proteins; reference to “a cell” includesmixtures of cells, and the like.

An “antisense strand” is a nucleic acid strand comprising a sequencecomplementary to that of a given mRNA, while a “sense strand” is anucleic acid strand comprising a sequence corresponding to that of themRNA.

“Attenuating” expression of a target gene refers to decreasing the levelof expression of the gene, e.g., as compared to the level in the absenceof a relevant interfering RNA.

A “caging group” is a moiety that can be employed to reversibly block,inhibit, or interfere with the activity (e.g., the biological activity)of a molecule (e.g., a polypeptide, a nucleic acid, a small molecule, adrug, etc.). The caging groups can, e.g., physically trap an activemolecule inside a framework formed by the caging groups. Typically,however, one or more caging groups are associated (covalently ornoncovalently) with the molecule but do not necessarily surround themolecule in a physical cage. For example, a single caging groupcovalently attached to an amino acid side chain required for thecatalytic activity of an enzyme can block the activity of the enzyme;the enzyme would thus be caged even though not physically surrounded bythe caging group. Caging groups can be, e.g., relatively small moietiessuch as carboxyl nitrobenzyl, 2-nitrobenzyl, nitroindoline,hydroxyphenacyl, DMNPE, or the like, or they can be, e.g., large bulkymoieties such as a protein or a bead. Caging groups can be removed froma molecule, or their interference with the molecule's activity can beotherwise reversed or reduced, by exposure to an appropriate type ofuncaging energy and/or exposure to an uncaging chemical, enzyme, or thelike.

A “photoactivatable” or “photoactivated” caging group is a caging groupwhose blockage, inhibition of, or interference with the activity of amolecule with which the photoactivatable caging group is associated canbe reversed or reduced by exposure to light of an appropriatewavelength. For example, exposure to light can disrupt a network ofcaging groups physically surrounding the molecule, reverse a noncovalentassociation with the molecule, trigger a conformational change thatrenders the molecule active even though still associated with the caginggroup, or cleave a photolabile covalent attachment to the molecule.

A “photolabile” caging group is one whose covalent attachment to amolecule is reversed (cleaved) by exposure to light of an appropriatewavelength. The photolabile caging group can be, e.g., a relativelysmall moiety such as carboxyl nitrobenzyl, 2-nitrobenzyl, nitroindoline,hydroxyphenacyl, DMNPE, or the like, or it can be, e.g., a relativelybulky group (e.g. a macromolecule, a protein) covalently attached to themolecule by a photolabile linker (e.g., a polypeptide linker comprisinga 2-nitrophenyl glycine residue).

A “cellular delivery module” or “cellular delivery agent” is a moietythat can mediate introduction into a cell of a molecule with which themodule is associated (covalently or noncovalently).

The term “eukaryote” refers to organisms belonging to the phylogeneticdomain Eucarya such as animals (e.g., mammals, insects, reptiles, birds,etc.), ciliates, plants, fungi (e.g., yeasts, etc.), flagellates,microsporidia, protists, etc. Additionally, the term “prokaryote” refersto non-eukaryotic organisms belonging to the Eubacteria (e.g.,Escherichia coli, Thermus thermophilus, etc.) and Archaea (e.g.,Methanococcus jannaschii, Methanobacterium thermoautotrophicum,Halobacterium species, etc.) phylogenetic domains.

“Expression of a gene” or “expression of a nucleic acid” meanstranscription of DNA into RNA (optionally including modification of theRNA, e.g., splicing) and/or translation of encoded RNA (e.g., mRNA) intoa polypeptide (possibly including subsequent modification of thepolypeptide, e.g., post-translational modification), as indicated by thecontext.

The term “gene” is used broadly to refer to any nucleic acid associatedwith a biological function. Genes typically include coding sequencesand/or the regulatory sequences required for expression of such codingsequences.

A “label” is a moiety that facilitates detection of a molecule. Commonlabels in the context of the present invention include fluorescent,luminescent, and/or colorimetric labels. Suitable labels includeradionuclides, enzymes, substrates, cofactors, inhibitors, fluorescentmoieties, chemiluminescent moieties, magnetic particles, and the like.Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241. Many labels are commercially available and can be used in thecontext of the invention.

The term “nucleic acid” encompasses any physical string of monomer unitsthat can be corresponded to a string of nucleotides, including a polymerof nucleotides (e.g., a typical DNA or RNA polymer), PNAs, modifiedoligonucleotides (e.g., oligonucleotides comprising nucleotides that arenot typical to biological RNA or DNA, such as 2′-O-methylatedoligonucleotides), and/or the like. A nucleic acid can be, e.g.,single-stranded or double-stranded. Unless otherwise indicated, aparticular nucleic acid sequence of this invention optionally comprisesor encodes complementary sequences, in addition to any sequenceexplicitly indicated. A nucleic acid (e.g., a polyribonucleotide, adouble-stranded RNA, or the like) of this invention is optionallynuclease resistant.

A nucleic acid that is “nuclease resistant” or “resistant to nucleaseactivity” is cleaved more slowly under typical reaction conditions for agiven nuclease (e.g., a 5′ to 3′ nuclease and/or an endonuclease) thanis a corresponding nucleic acid comprising only the four conventionaldeoxyribonucleotides (A, T, G, and/or C), or the four conventionalribonucleotides (U, A, G, and/or C), and phosphodiester linkages. Forexample, nucleic acids that incorporate 2′O methylated nucleotides aretypically more nuclease resistant than nucleic acids that incorporateonly conventional nucleotides. Many such modifications that impartnuclease resistance are known and can be adapted to the presentinvention.

An “oligonucleotide” or “polynucleotide” is a polymer comprising two ormore nucleotides. (For example, a “polyribonucleotide” is a polymercomprising two or more ribonucleotides.) The polymer can additionallycomprise non-nucleotide elements such as labels, quenchers, blockinggroups, or the like. The nucleotides of the oligonucleotide can bedeoxyribonucleotides, ribonucleotides and/or nucleotide analogs, can benatural or non-natural, and can be unsubstituted, unmodified,substituted or modified. The nucleotides can be linked by phosphodiesterbonds, or by phosphorothioate linkages, methylphosphonate linkages,boranophosphate linkages, or the like.

The “5′ end” of a polynucleotide refers to the nucleotide located at the5′ terminus of the polynucleotide. A moiety “attached at the 5′ end” ofthe polynucleotide can thus be attached to any part of the 5′ terminalnucleotide, e.g., the terminal 5′ phosphate or hydroxyl, the base, orthe ribose. Similarly, the “3′ end” of a polynucleotide refers to thenucleotide located at the 3′ terminus of the polynucleotide. A moiety“attached at the 3′ end” of the polynucleotide can thus be attached toany part of the 3′ terminal nucleotide, e.g., the terminal 3′ hydroxyl,the base, or the ribose.

A “subcellular delivery module” or “subcellular delivery agent” is amoiety that can mediate delivery and/or localization of an associatedmolecule to a particular subcellular location (e.g., a subcellularcompartment, a membrane, and/or neighboring a particular macromolecule).The subcellular delivery module can be covalently or noncovalentlyassociated with the molecule. Subcellular delivery modules include,e.g., peptide tags such as a nuclear localization signal ormitochondrial matrix-targeting signal.

A “synthetic oligonucleotide” or a “chemically synthesizedoligonucleotide” is an oligonucleotide made through in vitro chemicalsynthesis, as opposed to an oligonucleotide made either in vitro or invivo by a template-directed, enzyme-dependent reaction.

A “polypeptide” is a polymer comprising two or more amino acid residues(e.g., a peptide or a protein). The polymer can additionally comprisenon-amino acid elements such as labels, quenchers, blocking groups, orthe like and can optionally comprise modifications such as glycosylationor the like. The amino acid residues of the polypeptide can be naturalor non-natural and can be unsubstituted, unmodified, substituted ormodified.

A “protein transduction domain” is a polypeptide sequence that canmediate introduction of a covalently associated molecule into a cell.Protein transduction domains are typically short peptides (e.g., oftenless than about 16 residues). Example protein transduction domains havebeen derived from the HIV-1 protein Tat, the herpes simplex virusprotein VP22, and the Drosophila protein antennapedia; model proteintransduction domains have also been designed.

A “quencher” is a moiety that alters a property of a label (typically, afluorescent label) when it is in proximity to the label. The quenchercan actually quench an emission, but it does not have to, i.e., it cansimply alter some detectable property of the label, or, when proximal tothe label, cause a different detectable property than when not proximalto the label. A quencher can be e.g., an acceptor fluorophore thatoperates via energy transfer and re-emits the transferred energy aslight; other similar quenchers, called “dark quenchers,” do not re-emittransferred energy via fluorescence. A variety of labels and quenchersare found in Haughland (2003) Handbook of Fluorescent Probes andResearch Products Ninth Edition, available from Molecular Probes. Astraightforward discussion of FRET can be found in the Handbook at page25-26 and the references cited therein.

A “target mRNA” is an mRNA that is to be detected and/or whoseexpression is to be affected. A “target gene” is a gene whose expressionis to be detected (e.g., one or more corresponding mRNAs are to bedetected) and/or whose expression is to be affected. A target gene canbe, e.g., an endogenous gene or a heterologous gene (e.g., a geneintroduced into the cell through infection by a pathogen, or a geneintroduced through recombinant means). A target gene can be, e.g., aconstitutively expressed gene or an inducible gene; similarly, a targetmRNA can be, e.g., a constitutively expressed mRNA or an inducible mRNA.

“Uncaging energy” is energy that removes one or more caging groups froma caged molecule (or otherwise reverses the caging groups' blockage ofthe molecule's activity). Uncaging energy can be supplied, e.g., bylight, sonication, a heat source, a magnetic field, electromagneticradiation, or the like, as appropriate for the particular caginggroup(s).

The term “vector” refers to a means by which a nucleic acid can bepropagated and/or transferred between organisms, cells, or cellularcomponents. Vectors include plasmids, viruses, bacteriophage,pro-viruses, phagemids, transposons, and artificial chromosomes, and thelike, that replicate autonomously or can integrate into a chromosome ofa host cell. A vector can also be a naked RNA polynucleotide, a nakedDNA polynucleotide, a polynucleotide composed of both DNA and RNA withinthe same strand, a poly-lysine-conjugated DNA or RNA, apeptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like,that are not autonomously replicating. Most commonly, the vectors of thepresent invention are plasmids.

A variety of additional terms are defined or otherwise characterizedherein.

DETAILED DESCRIPTION

The term “RNA interference” (“RNAi,” sometimes called RNA-mediatedinterference, post-transcriptional gene silencing, or quelling) refersto a phenomenon in which the presence of double-stranded RNA in a cellresults in inhibition of expression of a gene comprising a sequenceidentical, or nearly identical, to that of the double-stranded RNA. Thedouble-stranded RNA responsible for inducing RNAi is called an“interfering RNA.” Expression of the gene is inhibited by the mechanismof RNAi as described below, in which the presence of the interfering RNAresults in degradation of mRNA transcribed from the gene and thus indecreased levels of the mRNA and any encoded protein.

The mechanism of RNAi has been and is being extensively investigated ina number of eukaryotic organisms and cell types. See, for example, thefollowing reviews: McManus and Sharp (2002) “Gene silencing in mammalsby small interfering RNAs” Nature Reviews Genetics 3:737-747; Hutvagnerand Zamore (2002) “RNAi: Nature abhors a double strand” Curr Opin Genet& Dev 200:225-232; Hannon (2002) “RNA interference” Nature 418:244-251;Agami (2002) “RNAi and related mechanisms and their potential use fortherapy” Curr Opin Chem Biol 6:829-834; Tuschl and Borkhardt (2002)“Small interfering RNAs: A revolutionary tool for the analysis of genefunction and gene therapy” Molecular Interventions 2:158-167; Nishikura(2001) “A short primer on RNAi: RNA-directed RNA polymerase acts as akey catalyst” Cell 107:415-418; and Zamore (2001) “RNA interference:Listening to the sound of silence” Nature Structural Biology 8:746-750.RNAi is also described in the patent literature; see, e.g., CA 2359180by Kreutzer and Limmer entitled “Method and medicament for inhibitingthe expression of a given gene”; WO 01/68836 by Beach et al. entitled“Methods and compositions for RNA interference”; WO 01/70949 by Grahamet al. entitled “Genetic silencing”; and WO 01/75164 by Tuschl et al.entitled “RNA sequence-specific mediators of RNA interference.”

In brief, double-stranded RNA introduced into a cell (e.g., into thecytoplasm) is processed, for example by an RNAse III-like enzyme calledDicer, into shorter double-stranded fragments called small interferingRNAs (siRNAs, also called short interfering RNAs). The length and natureof the siRNAs produced is dependent on the species of the cell, althoughtypically siRNAs are 21-25 nucleotides long (e.g., an siRNA may have a19 base pair duplex portion with two nucleotide 3′ overhangs at eachend). Similar siRNAs can be produced in vitro (e.g., by chemicalsynthesis or in vitro transcription) and introduced into the cell toinduce RNAi. The siRNA becomes associated with an RNA-induced silencingcomplex (RISC). Separation of the sense and antisense strands of thesiRNA, and interaction of the siRNA antisense strand with its targetmRNA through complementary base-pairing interactions, optionally occurs.Finally, the mRNA is cleaved and degraded.

Expression of a target gene in a cell can thus be specifically inhibitedby introducing an appropriately chosen double-stranded RNA into thecell. Guidelines for design of suitable interfering RNAs are known tothose of skill in the art. For example, interfering RNAs are typicallydesigned against exon sequences, rather than introns or untranslatedregions. Characteristics of high efficiency interfering RNAs may vary bycell type. For example, although siRNAs may require 3′ overhangs and 5′phosphates for most efficient induction of RNAi in Drosophila cells, inmammalian cells blunt ended siRNAs and/or RNAs lacking 5′ phosphates caninduce RNAi as effectively as siRNAs with 3′ overhangs and/or 5′phosphates (see, e.g., Czauderna et al. (2003) “Structural variationsand stabilizing modifications of synthetic siRNAs in mammalian cells”Nucl Acids Res 31:2705-2716). As another example, since double-strandedRNAs greater than 30-80 base pairs long activate the antiviralinterferon response in mammalian cells and result in non-specificsilencing, interfering RNAs for use in mammalian cells are typicallyless than 30 base pairs (for example, Caplen et al. (2001) “Specificinhibition of gene expression by small double-stranded RNAs ininvertebrate and vertebrate systems” Proc. Natl. Acad. Sci. USA98:9742-9747, Elbashir et al. (2001) “Duplexes of 21-nucleotide RNAsmediate RNA interference in cultured mammalian cells” Nature 411:494-498and Elbashir et al. (2002) “Analysis of gene function in somaticmammalian cells using small interfering RNAs” Methods 26:199-213describe the use of 21 nucleotide siRNAs to specifically inhibit geneexpression in mammalian cell lines, and Kim et al. (2005) “SyntheticdsRNA Dicer substrates enhance RNAi potency and efficacy” NatureBiotechnology 23:222-226 describes use of 25-30 nucleotide duplexes).The sense and antisense strands of a siRNA are typically, but notnecessarily, completely complementary to each other over thedouble-stranded region of the siRNA (excluding any overhangs). Theantisense strand is typically completely complementary to the targetmRNA over the same region, although some nucleotide substitutions can betolerated (e.g., a one or two nucleotide mismatch between the antisensestrand and the mRNA can still result in RNAi, although at reducedefficiency). The ends of the double-stranded region are typically moretolerant to substitution than the middle; for example, as little as 15bp (base pairs) of complementarity between the antisense strand and thetarget mRNA in the context of a 21 mer with a 19 bp double-strandedregion has been shown to result in a functional siRNA (see, e.g.,Czauderna et al. (2003) “Structural variations and stabilizingmodifications of synthetic siRNAs in mammalian cells” Nucl Acids Res31:2705-2716). Any overhangs can but need not be complementary to thetarget mRNA; for example, TT (two 2′-deoxythymidines) overhangs arefrequently used to reduce synthesis costs.

Although double-stranded RNAs (e.g., double-stranded siRNAs) wereinitially thought to be required to initiate RNAi, several recentreports indicate that the antisense strand of such siRNAs is sufficientto initiate RNAi. Single-stranded antisense siRNAs can initiate RNAithrough the same pathway as double-stranded siRNAs (as evidenced, forexample, by the appearance of specific mRNA endonucleolytic cleavagefragments). As for double-stranded interfering RNAs, characteristics ofhigh-efficiency single-stranded siRNAs may vary by cell type (e.g., a 5′phosphate may be required on the antisense strand for efficientinduction of RNAi in some cell types, while a free 5′ hydroxyl issufficient in other cell types capable of phosphorylating the hydroxyl).See, e.g., Martinez et al. (2002) “Single-stranded antisense siRNAsguide target RNA cleavage in RNAi” Cell 110:563-574; Amarzguioui et al.(2003) “Tolerance for mutations and chemical modifications in a siRNA”Nucl. Acids Res. 31:589-595; Holen et al. (2003) “Similar behavior ofsingle-strand and double-strand siRNAs suggests that they act through acommon RNAi pathway” Nucl. Acids Res. 31:2401-2407; and Schwarz et al.(2002) Mol. Cell 10:537-548.

Due to currently unexplained differences in efficiency between siRNAscorresponding to different regions of a given target mRNA, severalsiRNAs are typically designed and tested against the target mRNA todetermine which siRNA is most effective. Interfering RNAs can also beproduced as small hairpin RNAs (shRNAs, also called short hairpin RNAs),which are processed in the cell into siRNA-like molecules that initiateRNAi (see, e.g., Siolas et al. (2005) “Synthetic shRNAs as potent RNAitriggers” Nature Biotechnology 23:227-231).

The present invention provides a number of novel methods, compositions,and kits related to RNAi. For example, the invention provides methods inwhich a labeled interfering RNA is used as an in cell sensor to detectand/or quantitate a target mRNA. The labeled RNA includes a label whosesignal output changes when the labeled RNA initiates RNA interference(e.g., if the target mRNA is present in the cell). The labeled RNAsensor is optionally caged to permit temporal and/or spatial controlover activation of the sensor. Related kits, systems, and compositions(e.g., comprising labeled interfering RNAs for use as in cell sensors)are also provided.

As another example, the invention also provides caged interfering RNAs(e.g., photoactivatable interfering RNAs). Such a caged RNA includes,e.g., one or more caging groups that inhibit (e.g., prevent) the RNAfrom initiating RNA interference and whose removal or change inconformation permits the RNA to initiate RNA interference. Kits formaking the caged RNA and kits and systems comprising the caged RNA arealso features of the invention, as are methods of using a cagedinterfering RNA to selectively attenuate expression of a target gene ina cell. Using a caged interfering RNA in the methods permits theinitiation of RNAi to be precisely controlled, temporally and/orspatially.

As yet another example, the invention provides novel methods andcompositions for introducing interfering RNAs into cells. An interferingRNA can be covalently attached to a protein transduction domain and/orto a lipid (e.g., a myristoyl group) that can mediate its introductioninto a cell. Methods of introducing an interfering RNA into a cell, bycontacting the cell with the protein transduction domain- and/orlipid-linked interfering RNA, are also provided. The covalent attachmentbetween the protein transduction domain or lipid and the RNA isoptionally reversible (e.g., the attachment can be a photolabile linkeror a disulfide bond).

As yet another example, the invention provides additional methods ofselectively attenuating expression of a target gene in a cell. In someembodiments, transcription of an interfering RNA is controlled by use ofa caged activation component. In other embodiments, caged DNAs encodingan interfering RNA are introduced into a cell and then uncaged to permittranscription of the interfering RNA. The following sections describethe invention in more detail.

Related compositions, methods, systems, and kits involving RNAs capableof repressing translation of a target mRNA or silencing transcription ofa target gene are also described.

Interfering RNA Sensors

The use of double-stranded RNAs to attenuate expression of target genesby RNAi has been well described, including, e.g., the use of labels onsiRNAs to localize the siRNAs in transfected cells (e.g., to thecytoplasm, endosome, nucleus, or the like; see, e.g., Byrom et al.“Visualizing siRNA in mammalian cells: Fluorescence analysis of the RNAieffect” Ambion TechNotes 9(3)). Such labels are typically “nonfunctionallabels”; that is, they provide a signal output which remains constantregardless of whether the labeled siRNA initiates RNAi (e.g., interactswith RISC and/or the target mRNA). This invention, however, providesnovel methods of using labeled interfering RNAs as in cell sensors todetect expression of target genes. In the methods, the labels are“functional labels”; that is, the labels provide a signal output that isdependent on whether the labeled interfering RNA (e.g., the labeledsiRNA) initiates RNAi of the target mRNA (e.g., interacts with RISCand/or the target mRNA and undergoes strand separation, leading toendonucleolytic cleavage of the target mRNA). Compositions, systems, andkits related to the methods are also provided. The methods,compositions, systems, and kits overcome the above noted difficultiesassociated with current methods of detecting and/or quantitating mRNAtranscripts from cells, by enabling detection and/or quantitation ofmRNA in living cells and without requiring mRNA purification, reversetranscription, or cell lysis or fixation.

Methods

A first general class of embodiments provides methods of detecting atarget mRNA in a cell. In the methods, a labeled RNA is provided. Thelabeled RNA comprises an RNA comprising at least one double-strandedregion, the double-stranded region comprising a sense strand and anantisense strand, the antisense strand comprising a region which issubstantially complementary to a region of the target mRNA, and at leastone label. The labeled RNA is introduced into the cell, whereby thelabeled RNA initiates RNA interference of the target mRNA. This resultsin an initiation-dependent change in a signal output of the label. Thesignal output, which provides an indication of the presence of thetarget mRNA in the cell, is detected. The target mRNA can be, forexample, a constitutively expressed mRNA or an mRNA whose expression isinducible.

It is worth noting that initiation of RNAi of the target mRNA by thelabeled RNA can, but need not, result in a substantial attenuation ofexpression of the target mRNA. For example, expression of the targetmRNA can be unaffected, or expression of the target mRNA can bedecreased by at least about 0.001%, at least about 0.01%, at least about0.1%, at least about 1%, at least about 5%, at least about 10%, at leastabout 25%, at least about 50%, or at least about 75% or more, or caneven be reduced to an undetectable level.

In a preferred class of embodiments, the label is a fluorescent label,and the initiation-dependent change in the signal output of the label isa change in fluorescent emission. The methods can optionally be used toquantitate the amount of target mRNA present in the cell. For example,the intensity of the fluorescent emission can be measured. The intensityprovides an indication of the quantity of the target mRNA present in thecell.

In one class of embodiments, the labeled RNA also includes at least onequencher. The label and the quencher are positioned in the RNA such thatfluorescent emission by the label is quenched by the quencher.Initiation of RNA interference by the labeled RNA results in unquenchingof the label (and thus an increase in the fluorescent emission by thelabel). In this class of embodiments, the initiation-dependent change inthe signal output is thus an increase in the fluorescent emission by thelabel. For example, the label and quencher can be positioned on oppositestrands, in close enough proximity to each other that the label isquenched until the sense and antisense strands are separated.

In a related class of embodiments, the labeled RNA comprises twofluorescent labels, one being a donor and the other being an acceptor.The donor and acceptor are positioned within the RNA such that energytransfer (e.g., FRET) occurs between them (e.g., excitation of the donorresults in fluorescence by the acceptor). Initiation of RNA interferenceby the labeled RNA results in loss of energy transfer between the donorand the acceptor. This can be observed as an increase in fluorescence bythe donor or as a decrease in fluorescence by the acceptor. Thus, in apreferred class of embodiments, the initiation-dependent change in thesignal output is a decrease in fluorescent emission by the acceptorfollowing excitation of the donor. As in the preceding embodiments, thedonor and acceptor can, for example, be positioned on opposite strandsin close enough proximity to each other that energy transfer occursuntil the sense and antisense strands are separated.

The RNA can have any of a variety of structures, lengths, and/or thelike. Thus, in one class of embodiments, the RNA comprises a firstpolyribonucleotide comprising the sense strand and a secondpolyribonucleotide comprising the antisense strand. The RNA can be,e.g., a long double-stranded RNA that is cleaved by Dicer in the cell,or it can be, e.g., an siRNA. For example, the first polyribonucleotidecan comprise between 19 and 25 nucleotides, the secondpolyribonucleotide can comprise between 19 and 25 nucleotides, and thedouble-stranded region can comprise between 19 and 25 base pairs. Thefirst and second polyribonucleotides can form a duplex over their entirelength, or they can have overhangs (e.g., 5′ or 3′ overhangs; e.g., 21nt first and second polyribonucleotides can form a 19 bp double-strandedregion with 2 nucleotide overhangs, 23 nt polyribonucleotides can form a21 bp double-stranded region with 2 nucleotide overhangs, and so on).For example, in some embodiments, the first polyribonucleotide and thesecond polyribonucleotide each comprise a two nucleotide TT 3′ overhang(where T is 2′-deoxythymidine). The RNA is optionally nuclease resistantand optionally comprises one or more deoxyribonucleotides one or morePNA monomers, and/or one or more modified nucleotides (e.g., 2′-methylor 2′-O-allyl ribonucleotides) or internucleotide linkages (e.g.,phosphorothioate linkages).

As described in greater detail below, the RNA sensors can be, and inseveral embodiments are, caged. Thus, in some embodiments, at least onecaging group is associated with the RNA. For example, at least onecaging group can be covalently attached to a 5′ hydroxyl or a 5′phosphate of the second polyribonucleotide. Since this 5′ hydroxyl orphosphate is useful for an siRNA to initiate RNAi (Czauderna et al.(2003) “Structural variations and stabilizing modifications of syntheticsiRNAs in mammalian cells” Nucl Acids Res 31:2705-2716), caging the 5′hydroxyl or phosphate of the antisense strand permits the sensor to beuncaged and activated in a controlled manner.

In one class of embodiments, in which the first and secondpolyribonucleotides comprise 19-25 nt, the label is a fluorescent label,and the RNA further comprises at least one quencher. The label and thequencher are positioned in the RNA such that fluorescent emission by thelabel is quenched by the quencher, and initiation of RNA interference bythe labeled RNA results in unquenching of the label. Theinitiation-dependent change in the signal output is thus an increase inthe fluorescent emission by the label.

The label and quencher can be attached to a nucleic acid of theinvention at essentially any suitable position(s), e.g., at the 3′ end,at the 5′ end, and/or within either or both the first and secondpolyribonucleotides. For example, the label can be attached to the firstpolyribonucleotide and the quencher attached to the secondpolyribonucleotide, or the label can be attached to the secondpolyribonucleotide and the quencher attached to the firstpolyribonucleotide. For example, one of the label and the quencher canbe located within three nucleotides of the 3′ end of the firstpolyribonucleotide and the other of the label and the quencher can belocated within three nucleotides of the 3′ end of the secondpolyribonucleotide (see, e.g., FIG. 1 Panels A-C). As another example,one of the label and the quencher can be located within threenucleotides of the 3′ end of the first polyribonucleotide and the otherof the label and the quencher can be located within three nucleotides ofthe 5′ end of the second polyribonucleotide (see, e.g., FIG. 1 Panels Dand G). As yet another example, one of the label and the quencher can belocated within three nucleotides of the 5′ end of the firstpolyribonucleotide and the other of the label and the quencher can belocated within three nucleotides of the 3′ end of the secondpolyribonucleotide (see, e.g., FIG. 1 Panels E, F and H). As yet anotherexample, one of the label and the quencher can be located within threenucleotides of the 5′ end of the first polyribonucleotide and the otherof the label and the quencher can be located within three nucleotides ofthe 5′ end of the second polyribonucleotide (see, e.g., FIG. 1 Panel I).In other examples, the label and/or quencher can be in the middle of theRNA; e.g., one of the label and the quencher can be located more thanthree nucleotides from the 5′ end and more than three nucleotides fromthe 3′ end of the first polyribonucleotide, and the other of the labeland the quencher can be located more than three nucleotides from the 5′end and more than three nucleotides from the 3′ end of the secondpolyribonucleotide, or, one of the label and the quencher can be locatedwithin three nucleotides of the 5′ end or the 3′ end of the first orsecond polyribonucleotide and the other of the label and the quenchercan be located more than three nucleotides from the 5′ end and more thanthree nucleotides from the 3′ end of the opposite polyribonucleotide. Inone example embodiment, the label is attached at the 3′ end of the firstpolyribonucleotide and the quencher is attached at the 3′ end of thesecond polyribonucleotide. In a related example embodiment, the quencheris attached at the 3′ end of the first polyribonucleotide and the labelis attached at the 3′ end of the second polyribonucleotide. In yetanother example, one of the label and the quencher is attached at the 5′end of the first polyribonucleotide and the other of the label and thequencher is attached at the 3′ end of the second polyribonucleotide.

Techniques for determining and verifying suitable positions for thelabel and quencher are well known in the art. For example, the label andquencher are typically positioned such that they do not substantiallyreduce RNAi of the target mRNA as compared to an otherwise identical RNAlacking the label and quencher (e.g., the label and quencher preferablydo not interfere with siRNA binding to RISC, strand separation of thesiRNA, or binding of the antisense strand to the target mRNA). Forexample, if the quencher and label are located in the middle of thedouble-stranded region, the quencher can be attached to a nucleotide oneor more nucleotides removed from the complement of the nucleotide towhich the fluorescent label is attached. As another example, althoughoverhangs may not be necessary for siRNA function, 3′ and/or 5′overhangs of one, two, three, four, or more nucleotides can optionallybe used to position a quencher or label such that it does not interferewith RISC binding to the sensor (see, e.g., FIG. 1 panels G and H).

In a related class of embodiments in which the first and secondpolyribonucleotides comprise 19-25 nt, the labeled RNA comprises twofluorescent labels, one of which is a donor and the other of which is anacceptor. The donor and acceptor are positioned within the RNA such thatenergy transfer occurs between them. Initiation of RNA interference bythe labeled RNA results in loss of energy transfer between the donor andthe acceptor. The initiation-dependent change in signal output can thusbe, e.g., a decrease in fluorescent emission by the acceptor followingexcitation of the donor.

The donor and acceptor can be attached at essentially any suitablepositions, e.g., at the 3′ end, at the 5′ end, and/or within either orboth the first and second polyribonucleotides. For example, the donorcan be attached to the first polyribonucleotide and the acceptor to thesecond polyribonucleotide, or the donor can be attached to the secondpolyribonucleotide and the acceptor to the first polyribonucleotide. Forexample, one of the donor and the acceptor can be located within threenucleotides of the 3′ end of the first polyribonucleotide and the otherof the donor and the acceptor can be located within three nucleotides ofthe 3′ end of the second polyribonucleotide (see, e.g., FIG. 1 PanelsA-C). As another example, one of the donor and the acceptor can belocated within three nucleotides of the 3′ end of the firstpolyribonucleotide and the other of the donor and the acceptor can belocated within three nucleotides of the 5′ end of the secondpolyribonucleotide (see, e.g., FIG. 1 Panels D and G). As yet anotherexample, one of the donor and the acceptor can be located within threenucleotides of the 5′ end of the first polyribonucleotide and the otherof the donor and the acceptor can be located within three nucleotides ofthe 3′ end of the second polyribonucleotide (see, e.g., FIG. 1 Panel E,F and H). As yet another example, one of the donor and the acceptor canbe located within three nucleotides of the 5′ end of the firstpolyribonucleotide and the other of the donor and the acceptor can belocated within three nucleotides of the 5′ end of the secondpolyribonucleotide (see, e.g., FIG. 1 Panel I). In other examples, thedonor and/or acceptor can be in the middle of the RNA; e.g., one of thedonor and the acceptor can be located more than three nucleotides fromthe 5′ end and more than three nucleotides from the 3′ end of the firstpolyribonucleotide, and the other of the donor and the acceptor can belocated more than three nucleotides from the 5′ end and more than threenucleotides from the 3′ end of the second polyribonucleotide, or, one ofthe donor and the acceptor can be located within three nucleotides ofthe 5′ end or the 3′ end of the first or second polyribonucleotide andthe other of the donor and the acceptor can be located more than threenucleotides from the 5′ end and more than three nucleotides from the 3′end of the opposite polyribonucleotide. In one example embodiment, thedonor is attached at the 3′ end of the first polyribonucleotide and theacceptor is attached at the 3′ end of the second polyribonucleotide. Ina related example embodiment, the acceptor is attached at the 3′ end ofthe first polyribonucleotide and the donor is attached at the 3′ end ofthe second polyribonucleotide. In yet another example, one of the donorand the acceptor is attached at the 5′ end of the firstpolyribonucleotide and the other of the donor and the acceptor isattached at the 3′ end of the second polyribonucleotide. Techniques fordetermining and verifying suitable positions for the donor and acceptorare well known in the art.

Instead of comprising two polyribonucleotides, in some embodiments, theRNA of interest comprises a self-complementary polyribonucleotide (e.g.,a shRNA). Label/quencher or acceptor/donor combinations can be similarlypositioned within the self-complementary polyribonucleotide.

As noted, the length of the RNA can vary. For example, thedouble-stranded region can comprise fewer than about 1500 base pairs,fewer than about 1000 base pairs, fewer than about 500 base pairs, fewerthan about 250 base pairs, fewer than about 150 base pairs, fewer thanabout 80 base pairs, fewer than about 50 base pairs, fewer than about 30base pairs, or fewer than about 25 base pairs.

As noted, the RNA sensors can optionally be caged. Caging a sensor,e.g., with a photolabile group, allows the initiation of RNAi, and thusthe detection of the target mRNA, to be precisely controlled, temporallyand/or spatially. This provides a number of advantages. For example, acaged RNA sensor can be introduced into a cell, e.g., by lipofection.The cell can be permitted to recover from the manipulations necessary tointroduce the sensor before uncaging of the sensor permits initiation ofRNAi and detection of the target transcript. As another example, untilthe sensor is uncaged, the interfering RNA exerts no effect on the cell.As yet another example, caging the interfering RNA can protect it fromnucleases and thus extend its half-life.

Thus, in one class of embodiments, the labeled RNA further comprises oneor more first caging groups associated with the RNA. The first caginggroups inhibit the RNA from initiating RNA interference of the targetmRNA in the cell. RNA interference of the target mRNA is initiated byexposing the cell to uncaging energy of a first type, whereby exposureto the uncaging energy frees the RNA from inhibition by the first caginggroups.

The first caging groups can inhibit the RNA from initiating RNAinterference of the target mRNA by at least about 25%, at least about30%, at least about 35%, at least about 50%, at least about 75%, atleast about 90%, at least about 95%, or at least about 98%, as comparedto the RNA in the absence of the first caging groups. For example, ifintroduction of an siRNA into a cell decreases expression of its targetmRNA to 10% of normal (i.e., expression in a cell not comprising thesiRNA), then introduction of the corresponding caged siRNA into a cellwould decrease expression to 55% of normal if the caging groups inhibitthe RNA from initiating RNA interference by 50% (under equivalentconditions). In one class of embodiments, the first caging groupsprevent the RNA from initiating RNA interference of the target mRNA(i.e., introduction of the caged RNA into a cell has no effect onexpression of the target mRNA). Removal of or an induced conformationalchange in the first caging groups typically permits the RNA to initiateRNA interference of the target mRNA.

The one or more first caging groups associated with the RNA can becovalently attached to or non-covalently associated with the RNA. See,e.g., FIGS. 5 and 6 for a few of the possible examples of sites ofattachment of the caging groups (e.g., at one or more bases, riboses,phosphate groups and/or terminal hydroxyls, within and/or at the end ofeither or both strands of the RNA). In one embodiment, the RNA comprisesa first polyribonucleotide comprising the sense strand and a secondpolyribonucleotide comprising the antisense strand, and the first caginggroup is covalently attached to the first polyribonucleotide and to thesecond polyribonucleotide. For example, the first caging group can beattached to the 5′ end of the first polyribonucleotide and to the 3′ endof the second polyribonucleotide, or, preferably, it can be attached tothe 3′ end of the first polyribonucleotide and to the 5′ end of thesecond polyribonucleotide (FIG. 4). The caging group linking the twopolyribonucleotides can, for example, be photolabile.

In a preferred aspect, the one or more first caging groups arephotoactivatable (e.g., photolabile). Thus, in a preferred class ofembodiments, exposing the cell to uncaging energy of the first typecomprises exposing the cell to light of a first wavelength (e.g., lightwith a wavelength between about 60 nm and about 400 nm, between about400 nm and about 700 nm, and/or between about 700 nm and about 1000 nm).Other caging groups are removable via input of different uncagingenergies, e.g., the one or more caging groups can be removable bysonication or application of heat, or can be removed by a chemical orenzyme.

Exposing the cell to light of a first wavelength optionally comprisesexposing the cell to light such that the intensity of the light and theduration of exposure to the light are controlled such that a firstportion (which can be a selected amount) of the caged labeled RNA isuncaged and a second portion of the caged labeled RNA remains caged. Putanother way, the uncaging rate can be controlled. Furthermore, theuncaging step can be repeated until the caged RNA is depleted.

As noted, caging permits temporal control over activation of the sensor.For example, the method can include stimulating the cell and uncagingthe sensor at a preselected time with respect to the stimulus. Forexample, the method can include contacting the cell and a test compoundand exposing the cell to the uncaging energy at a preselected time pointwith respect to a time at which the cell and the test compound arecontacted (e.g., to determine if the test compound directly orindirectly affects expression of the target mRNA). Caging also permitsspatial control over activation of the sensor. For example, the uncagingenergy can be directed at a preselected subset of a cell populationcomprising the cell.

Various techniques (e.g., lipofection, microinjection, orelectroporation) can be used to introduce the labeled RNA into the cell.In one class of embodiments, the labeled RNA also includes a cellulardelivery module, associated with the RNA, that can mediate introductionof the labeled RNA into the cell. In this class of embodiments,introducing the labeled RNA into the cell comprises contacting the cellwith the labeled RNA associated with the cellular delivery module.

The cellular delivery module optionally comprises a polypeptide, e.g., aPEP-1 peptide or an amphipathic peptide (e.g., an MPG or an MPG^(ΔNLS)peptide; see Simeoni et al. (2003) “Insight into the mechanism of thepeptide-based gene delivery system MPG: Implications for delivery ofsiRNA into mammalian cells” Nucl Acids Res 31: 2717-2724), covalently ornoncovalently associated with the RNA. As another example, thepolypeptide can be a cationic peptide (e.g., a homopolymer of histidine,lysine, or D-arginine) that is covalently or noncovalently (e.g., byelectrostatic interaction with the negatively charged RNA) associatedwith the RNA. In one class of embodiments, the cellular delivery modulecomprises a protein transduction domain, e.g., derived from an HIV-1 Tatprotein, from a herpes simplex virus VP22 protein, or from a Drosophilaantennapedia protein (e.g., Penetratin™). In one aspect, the proteintransduction domain is a model protein transduction domain, e.g., ahomopolymer of D-arginine, e.g., 8-D-Arg. The protein transductiondomain can be covalently attached directly to the RNA, or can beindirectly associated with the RNA (for example, the proteintransduction domain can be covalently coupled to a bead or to a carrierprotein such as BSA, which is in turn coupled to the RNA, e.g., througha photolabile or cleavable linker; e.g., FIGS. 8-9).

The cellular delivery module can be noncovalently associated with theRNA, or the cellular delivery module can be covalently attached to theRNA. For example, the covalent attachment between the cellular deliverymodule and the RNA is optionally reversible by exposure to light of apreselected wavelength, and the method includes exposing the cell tolight of the preselected wavelength. As another example, the covalentattachment is optionally a disulfide bond or an ester linkage that isreduced or cleaved once the sensor is inside the cell.

In certain embodiments, the cellular delivery module can also serve as acaging group. For example, the RNA can comprise a firstpolyribonucleotide comprising the sense strand and a secondpolyribonucleotide comprising the antisense strand, and the cellulardelivery module can be covalently attached to the firstpolyribonucleotide and/or to the second polyribonucleotide (e.g., by aphotolabile linker; see, e.g., FIGS. 7-10). The cellular delivery modulecan mediate introduction of the RNA into the cell, where the presence ofthe cellular delivery module prevents the RNA from initiating RNAinterference until the cellular delivery module is removed (e.g., byexposing the cell to light of an appropriate wavelength to cleave thephotolabile linker).

Optionally, the cellular delivery module covalently attached to the RNAcomprises a lipid, e.g., a fatty acid. For example, the RNA can becovalently attached to one or more myristoyl groups, e.g., via aphotolabile linker (FIG. 10).

In one aspect, the cellular delivery module is associated with one ormore second caging groups, which inhibit (e.g., prevent) the cellulardelivery module from mediating introduction of the labeled RNA into thecell. In this aspect, the method includes initiating introduction of thelabeled RNA into the cell by exposing the labeled RNA to uncaging energyof a second type (which is typically different from the uncaging energyof the first type if first caging groups are present on the RNA),whereby exposure to the uncaging energy frees the cellular deliverymodule from inhibition by the second caging groups.

The methods can optionally be used to monitor gene expression, forexample, induction of transcription of the target mRNA in response to astimulus. Thus, in one class of embodiments, the methods includestimulating the cell, e.g., by adding a test compound (e.g., a drug, acandidate drug, a receptor agonist or putative agonist, or the like), bychanging growth conditions, by adding other cells, etc.

The methods can be used to examine expression of the target mRNA, e.g.,in two different cell populations, one stimulated and one not.Similarly, expression of the target mRNA can be monitored in a singlecell (or a single cell population) before and after stimulation of thecell. Thus, in one embodiment, the signal output is detected at aplurality of time points with respect to a time at which the cell isstimulated.

The methods optionally include introducing a plurality of RNA sensors(labeled interfering RNAs) into the cell to simultaneously monitorexpression of a plurality of target mRNAs. The labels on the differentRNAs typically have detectably different signal outputs. For example,the different RNAs can comprise different fluorescent label/quenchercombinations (see, e.g., FIG. 2) or different donor/acceptorcombinations or a combination of FRET, fluorophore/quencher, and TR-FRETsensors can be used (see, e.g., FIG. 3). The different sensors areoptionally caged, e.g., with photolabile caging groups removable bydifferent wavelengths of light, such that the different sensors can beuncaged at different time points.

In a related class of embodiments, a reference sensor is also introducedinto the cell. Signal output from the target sensor's label (indicatingthe presence of the target mRNA, e.g., an inducible mRNA, in the cell)can be normalized by comparison with a signal output from the referencesensor. Such a reference sensor can comprise a labeled interfering RNAagainst a constitutively expressed or housekeeping gene (e.g., GAPDH,actin, or the like).

In one class of embodiments, the methods are used to determine howefficiently the RNA attenuates (or knocks-down) expression of the targetmRNA. In these embodiments, an intensity of the signal output (e.g.,intensity of a fluorescent emission) is measured. The intensity providesan indication of the quantity (e.g., relative or absolute quantity) ofthe target mRNA present in the cell, which provides an indication of theefficiency with which the labeled RNA reduces expression of the targetmRNA.

Kits

Another aspect of the invention includes kits related to the methods.For example, one class of embodiments provides a kit for detecting atarget mRNA in a cell. The kit includes a labeled RNA and instructionsfor using the labeled RNA to detect the presence of the target mRNA inthe cell, packaged in one or more containers. The labeled RNA comprisesan RNA comprising at least one double-stranded region, thedouble-stranded region comprising a sense strand and an antisensestrand, the antisense strand comprising a region which is substantiallycomplementary to a region of the target mRNA. The labeled RNA alsocomprises at least one label, wherein initiation of RNA interference ofthe target mRNA by the labeled RNA in the cell results in aninitiation-dependent change in a signal output of the label.

The instructions can include, for example, instructions for introducingthe labeled RNA into the cell, detecting a fluorescent signal from theRNA, interpreting the fluorescent signal (including quantitating themRNA based on the intensity of the fluorescent signal), and the like.

All of the various optional configurations and features noted for theembodiments above apply here as well, to the extent they are relevant,e.g., for label configurations (e.g., use of fluorescent labels,fluorescent label/quencher, and donor/acceptor combinations), signaloutput types, RNA configurations (e.g., one or two polyribonucleotides,of various lengths, with or without overhangs, etc.), use of caginggroups (e.g., photolabile caging groups), appropriate uncaging energies(light, heat, sonic, etc.), use of cellular delivery modules (e.g.,amphipathic peptides, cationic peptides, protein transduction domains,and lipids), and the like.

In addition, it is worth noting that the kit optionally also includes atleast one buffer and/or at least one delivery reagent. The deliveryreagent can be essentially any reagent that can mediate introduction ofthe labeled RNA into the cell; for example, the delivery reagent cancomprise a polypeptide (e.g., a PEP-1 peptide, an amphipathic peptide,e.g., an MPG or MPG^(ΔNLS) peptide, or a cationic peptide, e.g.,poly-His, poly-Lys, or poly-D-Arg) or at least one lipid (e.g., a lipidoptimized for lipofection of the given labeled RNA, or a lipidcomprising at least one myristoyl group to be covalently attached to thelabeled RNA). In one class of embodiments, the labeled RNA is caged. Inthis class of embodiments, the kit optionally includes a control reagentfor monitoring uncaging efficiency (e.g., a caged fluorophore, e.g.,caged FITC) and/or an uncaged version of the labeled RNA (e.g., to beused as a control to monitor uncaging of the caged labeled RNA, maximalknock down of the target mRNA, and/or the like). The kit also optionallyincludes packaging or instructional materials for such additionalreagents.

An additional class of embodiments also provides a kit for detecting atarget mRNA in a cell. In this class of embodiments, the kit comprises atarget RNA sensor and a reference RNA sensor, packaged in one or morecontainers. The target RNA sensor comprises a first RNA comprising atleast one double-stranded region, the double-stranded region comprisinga sense strand and an antisense strand, the antisense strand comprisinga region which is substantially complementary to a region of a targetmRNA, and at least one first label, wherein initiation of RNAinterference of the target mRNA by the first RNA in the cell results inan initiation-dependent change in a signal output of the first label.The reference RNA sensor comprises a second RNA comprising at least onedouble-stranded region, the double-stranded region comprising a sensestrand and an antisense strand, the antisense strand comprising a regionwhich is substantially complementary to a region of a reference mRNA,and at least one second label, wherein initiation of RNA interference ofthe reference mRNA by the second RNA in the cell results in aninitiation-dependent change in a signal output of the second label;packaged in one or more containers. Typically, the signal output of thefirst label is detectably different from the signal output of the secondlabel.

The target and reference mRNAs can be essentially any mRNAs. Forexample, the target mRNA can be an inducible mRNA while the referencemRNA is a constitutively expressed or housekeeping mRNA (e.g., GAPDH,actin, or the like).

All of the various optional configurations and features noted for theembodiments above apply here as well, to the extent they are relevant,e.g., for label configurations (e.g., use of fluorescent labels,fluorescent label/quencher, and donor/acceptor combinations), signaloutput types, RNA configurations (e.g., one or two polyribonucleotides,of various lengths, with or without overhangs, etc.), use of caginggroups (e.g., photolabile caging groups), appropriate uncaging energies(light, heat, sonic, etc.), use of cellular delivery modules (e.g.,amphipathic peptides, protein transduction domains, and lipids), and thelike.

In addition, it is worth noting that the kit optionally also includesone or more of: instructions (e.g., for using the target and referenceRNA sensors to detect the presence of the target mRNA in the cell and/orfor using the target and reference RNA sensors to quantitate an amountof the target mRNA present in the cell), a buffer, or a delivery reagentwhich can mediate introduction of the labeled RNA into the cell (forexample, a polypeptide, e.g., a PEP-1 peptide, an amphipathic peptide,e.g., an MPG or MPG^(ΔNLS) peptide, or a cationic peptide, or at leastone lipid, e.g., a lipid optimized for lipofection of the given labeledRNA or a lipid comprising at least one myristoyl group to be covalentlyattached to the labeled RNA).

Compositions

Yet another aspect of the invention provides compositions related to themethods (e.g., compositions produced by the methods or facilitating useof the methods). For example, one class of embodiments provides acomposition comprising a population of labeled RNAs (e.g., identicallabeled RNAs) for detecting a target mRNA in a cell. The target mRNAand/or the cell are also optionally features of the composition. Eachlabeled RNA comprises an RNA comprising at least one double-strandedregion, the double-stranded region comprising a sense strand and anantisense strand, the antisense strand comprising a region which issubstantially complementary to a region of the target mRNA, and at leastone label. The label is located a preselected position in the RNA, andinitiation of RNA interference of the target mRNA by the labeled RNA inthe cell results in an initiation-dependent change in a signal output ofthe label.

All of the various optional configurations and features noted for theembodiments above apply here as well, to the extent they are relevant,e.g., for label configurations (e.g., use of fluorescent labels,fluorescent label/quencher, and donor/acceptor combinations), signaloutput types, RNA configurations (e.g., one or two polyribonucleotides,of various lengths, with or without overhangs, etc.), use of caginggroups (e.g., photolabile caging groups), appropriate uncaging energies(light, heat, sonic, etc.), use of cellular delivery modules (e.g.,amphipathic peptides, protein transduction domains, and lipids), and thelike. It is worth noting that a quencher or a second label, if presentin the RNA, is optionally also located a preselected position in theRNA. The composition comprising the population optionally also includesthe target mRNA and/or a cell, e.g., a cell comprising the populationand/or the target mRNA.

Using a population of RNA sensors in which the label is located at apreselected position in the RNA has several advantages over usinglabeled RNAs where the labels are located at random positions in theRNA. For example, the preselected label position can be chosen such thatthe initiation-dependent change in the signal output of the label ismaximized (e.g., label and quencher or acceptor and donor positions canbe selected to maximize the change in signal output). As anotherexample, the preselected label position can be chosen such that thepresence of the label does not substantially interfere with initiationof RNAi by the labeled RNA, whereas random labeling of an interferingRNA can result in labels being attached at positions where theyinterfere with RISC binding to the labeled RNA or the like.

In another class of embodiments, the invention provides a compositioncomprising a target RNA sensor and a reference RNA sensor. The targetRNA sensor comprises a first RNA comprising at least one double-strandedregion, the double-stranded region comprising a sense strand and anantisense strand, the antisense strand comprising a region which issubstantially complementary to a region of a target mRNA, and at leastone first label, wherein initiation of RNA interference of the targetmRNA by the first RNA in the cell results in an initiation-dependentchange in a signal output of the first label. The reference RNA sensorcomprises a second RNA comprising at least one double-stranded region,the double-stranded region comprising a sense strand and an antisensestrand, the antisense strand comprising a region which is substantiallycomplementary to a region of a reference mRNA, and at least one secondlabel, wherein initiation of RNA interference of the reference mRNA bythe second RNA in the cell results in an initiation-dependent change ina signal output of the second label. Typically, the signal output of thefirst label is detectably different from the signal output of the secondlabel.

The target and reference mRNAs can be essentially any mRNAs. Forexample, the target mRNA can be an inducible mRNA while the referencemRNA is a constitutively expressed or housekeeping mRNA (e.g., GAPDH,actin, or the like).

All of the various optional configurations and features noted for theembodiments above apply here as well, to the extent they are relevant,e.g., for label configurations (e.g., use of fluorescent labels,fluorescent label/quencher, and donor/acceptor combinations), signaloutput types, RNA configurations (e.g., one or two polyribonucleotides,of various lengths, with or without overhangs, etc.), use of caginggroups (e.g., photolabile caging groups), appropriate uncaging energies(light, heat, sonic, etc.), use of cellular delivery modules (e.g.,amphipathic peptides, protein transduction domains, and lipids), and thelike. It is worth noting that the composition optionally also includesthe target mRNA, the reference mRNAs and/or a cell, e.g., a cellcomprising the target and reference sensors and/or the target mRNA.

Systems

In another aspect, systems and/or apparatus comprising the compositions(e.g., the labeled RNAs, cells comprising the labeled RNAs, or the like)noted above and, e.g., components such as detectors, fluid handlingapparatus, sources of uncaging energy, or the like, are a feature of theinvention.

In general, various automated systems can be used to perform some or allof the method steps as noted herein. In addition to practicing some orall of the method steps herein, digital or analog systems, e.g.,comprising a digital or analog computer, can also control a variety ofother functions such as a user viewable display (e.g., to permit viewingof method results by a user) and/or control of output features.

For example, certain of the methods described above are optionallyimplemented via a computer program or programs (e.g., that perform orassist in detection of target mRNA). Thus, the present inventionprovides digital systems, e.g., computers, computer readable media,and/or integrated systems comprising instructions (e.g., embodied inappropriate software) for performing the methods herein. For example, adigital system comprising instructions for interpreting the change insignal output from the label to determine whether the target mRNA ispresent in the cell and/or to determine the quantity of the target mRNApresent in the cell, as described herein, is a feature of the invention.The digital system can also include information (data) corresponding tosignal output intensities or the like. The system can also aid a user inperforming mRNA detection according to the methods herein, or cancontrol laboratory equipment which automates introduction of the labeledRNAs into the cells, detection of the signal outputs, or the like.

Standard desktop applications such as word processing software (e.g.,Microsoft Word™ or Corel WordPerfect™) and/or database software (e.g.,spreadsheet software such as Microsoft Excel™, Corel Quattro Pro™, ordatabase programs such as Microsoft Access™ or Paradox™) can be adaptedto the present invention by inputting data which is loaded into thememory of a digital system and performing an operation as noted hereinon the data. For example, systems can include the foregoing softwarehaving the appropriate signal intensity (e.g., fluorescent intensity)data, etc., e.g., used in conjunction with a user interface (e.g., a GUIin a standard operating system such as a Windows, Macintosh or LINUXsystem) to perform any analysis noted herein, or simply to acquire data(e.g., in a spreadsheet) to be used in the methods herein.

Systems typically include, e.g., a digital computer with software forperforming signal output interpretation and/or mRNA quantitation, or thelike, as well as data sets entered into the software system comprisingsignal output intensities or the like. The computer can be, e.g., a PC(Intel x86 or Pentium chip-compatible DOS,™ OS2,™ WINDOWS,™ WINDOWS NT,™WINDOWS95,™ WINDOWS98,™ LINUX, Apple-compatible, MACINTOSH™ compatible,Power PC compatible, or a UNIX compatible (e.g., SUN™ work station)machine) or other commercially common computer which is known to one ofskill. Software for performing analysis of signal output and/or mRNAquantitation can be constructed by one of skill using a standardprogramming language such as Visualbasic, Fortran, Basic, Java, or thelike, according to the methods herein.

Any system controller or computer optionally includes a monitor whichcan include, e.g., a cathode ray tube (“CRT”) display, a flat paneldisplay (e.g., active matrix liquid crystal display, liquid crystaldisplay), or others. Computer circuitry is often placed in a box whichincludes numerous integrated circuit chips, such as a microprocessor,memory, interface circuits, and others. The box also optionally includesa hard disk drive, a floppy disk drive, a high capacity removable drivesuch as a writeable CD-ROM, and other common peripheral elements.Inputting devices such as a keyboard or mouse optionally provide forinput from a user and for user selection of the wavelength offluorescent emission to be monitored, or the like, in the relevantcomputer system.

The computer typically includes appropriate software for receiving userinstructions, either in the form of user input into a set parameterfields, e.g., in a GUI, or in the form of preprogrammed instructions,e.g., preprogrammed for a variety of different specific operations. Thesoftware then converts these instructions to appropriate language forinstructing the system to carry out any desired operation. For example,in addition to performing signal output analysis, a digital system cancontrol laboratory equipment for liquid handling, signal detection, orthe like according to the relevant method herein.

The invention can also be embodied within the circuitry of anapplication specific integrated circuit (ASIC) or programmable logicdevice (PLD). In such a case, the invention is embodied in a computerreadable descriptor language that can be used to create an ASIC or PLD.The invention can also be embodied within the circuitry or logicprocessors of a variety of other digital apparatus, such as PDAs, laptopcomputer systems, displays, image editing equipment, etc.

Applications

The methods, compositions, systems, and kits described above have anumber of applications. For example, as noted, labeled interfering RNAsensors can be used to detect the presence of a target mRNA, toquantitate an amount of a target mRNA present in a cell, and/or todetect activation of a target gene. Additional applications include, butare not limited to, detection of specific biological activities (e.g.,GCPR activation, disease, cell migration, cell death, and the like) viadetection of activation of biomarker target genes; detection of splicevariants of mRNAs, e.g., as biomarkers of biological activities;detection of drug effects via detection of activation of biomarkertarget genes; and performance of ADME (Absorption, Distribution,Metabolism and Excretion) toxicity assays via detection of activation ofbiomarker target genes. If desired, high throughput cell-based assaysusing labeled interfering RNA sensors can be designed for the aboveexample applications.

Caged Interfering RNA

In one aspect of this invention, caging groups (e.g., photo-labilecaging groups) are used to precisely control the timing and/or locationof RNA interference. For example, this invention featuresphotoactivatable (photo activated, PA) interfering RNA sensors suitablefor monitoring transcript expression in cells; the sensors are designedfor simple operation and are suitable for use in a wide array ofinstruments (e.g., fluorescent instrument platforms). The caged (e.g.,PA) interfering RNAs and methods of use thereof described herein aresuitable for applications in, e.g., clinical and basic research and drugdiscovery.

The advantages of using a caged (e.g., PA) reaction format include: (1.)controlled activation of reaction components (e.g., controlledinitiation of RNAi), (2.) improved assay precision, achieved e.g., (a.)by reducing number of fluidic handling steps in HTS assays—reducing thenumber of steps (each additional pipetting step can introduce more errorinto an assay) and/or (b.) by facilitating simultaneous activation oflarge numbers of assays within millisecond, and (3.) simplifiedautomation and design of miniaturized platforms by reducing the numberof steps required. Finally, the caged (e.g., PA) reaction format permitsspecific activation of a reaction in specific locations—e.g., a subsetof wells or locations within a microarray, microfluidic device and/orother miniaturized formats, or even within an organism (e.g., activationof specific locations separated by no more than about a micron ispossible).

An additional advantage of using caged compounds in cells is that caginga molecule frequently renders the molecule more resistant to nucleases,proteases, lipases, and the like, thus extending its half-life in thecell. Caging a molecule which already possesses enhanced resistance todegradation (e.g., by a nuclease or protease, e.g., by incorporation ofunnatural amino acids and/or nucleotides into the molecule) offerssimilar advantages in terms of molecule half-life in the cell or lysate(e.g., thus minimizing false-positive results from undesirable cleavageof a FRET-based sensor or probe).

Yet another advantage of using caged compounds (e.g., caged interferingRNAs) in cells is that caging a toxic molecule (e.g., an interfering RNAagainst an essential gene) frequently protects the cell from the effectof the molecule. This permits compounds that might otherwise be toodisruptive to the cell to be utilized in in-cell assays; the cell is notsubject to the adverse effect of the compound until the compound isuncaged during the assay.

Compositions

One general class of embodiments provides a composition comprising acaged interfering RNA. The caged RNA includes an RNA having at least onedouble-stranded region, the double-stranded region comprising a sensestrand and an antisense strand, the antisense strand comprising a regionwhich is substantially complementary to a region of a target mRNA. Thecaged RNA also includes one or more first caging groups associated withthe RNA. The first caging groups inhibit (e.g., prevent) the RNA frominitiating RNA interference of the target mRNA in a cell comprising thecaged RNA.

The first caging groups can inhibit the RNA from initiating RNAinterference of the target mRNA by at least about 25%, at least about30%, at least about 35%, at least about 50%, at least about 75%, atleast about 90%, at least about 95%, or at least about 98%, as comparedto the RNA in the absence of the first caging groups. In one class ofembodiments, the first caging groups prevent the RNA from initiating RNAinterference of the target mRNA (i.e., introduction of the caged RNAinto a cell has no effect on expression of the target mRNA). Removal ofor an induced conformational change in the first caging groups typicallypermits the RNA to initiate RNA interference of the target mRNA.

Note that the first caging groups “inhibiting the RNA from initiatingRNA interference” is not meant to imply that the first caging groupsinhibit any particular step(s) in the RNAi pathway. For example, thefirst caging groups can interfere with phosphorylation of a 5′ hydroxylof the antisense strand, RISC binding to the interfering RNA, strandseparation of the sense and antisense strands of the interfering RNA,and/or any other step in the RNAi pathway leading to cleavage anddegradation of the target mRNA. Initiation of RNAi by the RNA can beindicated, for example, by a decrease in the expression levels of thetarget mRNA and/or appearance of specific endonucleolytic cleavagefragments of the target mRNA, as is known in the art.

It is also worth noting that initiation of RNAi of the target mRNA bythe RNA need not, but typically does, result in a substantialattenuation of expression of the target mRNA. For example, expression ofthe target mRNA can be unaffected, or expression of the target mRNA canbe decreased by at least about 0.001%, at least about 0.01%, at leastabout 0.1%, at least about 1%, at least about 5%, at least about 10%, orpreferably at least about 25%, at least about 50%, or at least about 75%or more, or can even be reduced to an undetectable level.

The RNA can have any of a variety of structures, lengths, and/or thelike. Thus, in one class of embodiments, the RNA comprises a firstpolyribonucleotide comprising the sense strand and a secondpolyribonucleotide comprising the antisense strand. The RNA can be,e.g., a long double-stranded RNA that is cleaved by Dicer in the cell,or it can be, e.g., an siRNA. For example, the first polyribonucleotidecan comprise between 19 and 25 nucleotides, the secondpolyribonucleotide can comprise between 19 and 25 nucleotides, and thedouble-stranded region can comprise between 19 and 25 base pairs. Thefirst and second polyribonucleotides can form a duplex over their entirelength, or they can have overhangs (e.g., 5′ or 3′ overhangs; e.g., 21nt first and second polyribonucleotides can form a 19 bp double-strandedregion with 2 nucleotide overhangs, 23 nt polyribonucleotides can form a21 bp double-stranded region with 2 nucleotide overhangs, and so on).For example, in some embodiments, the first polyribonucleotide and thesecond polyribonucleotide each comprise a two nucleotide TT 3′ overhang(where T is 2′-deoxythymidine). As another example, the first and secondpolyribonucleotides can each comprise between 25 and 30 nucleotides(e.g., 27 nucleotides) and form a duplex over their entire length. TheRNA is optionally nuclease resistant and optionally comprises one ormore deoxyribonucleotides, one or more PNA monomers, and/or one or moremodified nucleotides (e.g., 2′-methyl or 2′-O-allyl ribonucleotides) orinternucleotide linkages (e.g., phosphorothioate linkages). In oneembodiment, at least one of the one or more first caging groups iscovalently attached to a 5′ hydroxyl or a 5′ phosphate of the secondpolyribonucleotide. Since this 5′ hydroxyl or phosphate is useful for ansiRNA to initiate RNAi, caging the 5′ hydroxyl or phosphate of theantisense strand permits the RNA to be uncaged and activated in acontrolled manner.

As noted, in certain embodiments, the RNA comprises a firstpolyribonucleotide comprising the sense strand and a secondpolyribonucleotide comprising the antisense strand. In otherembodiments, the RNA comprises a self-complementary polyribonucleotide(e.g., a hairpin, a shRNA). In either case, the double-stranded regionoptionally comprises fewer than about 25 base pairs, fewer than about 30base pairs, fewer than about 50 base pairs, fewer than about 80 basepairs, fewer than about 150 base pairs, fewer than about 250 base pairs,fewer than about 500 base pairs, fewer than about 1000 base pairs, orfewer than about 1500 base pairs. Although a double-stranded regioncomprising about 19-25 base pairs is typically sufficient to initiateRNAi, longer regions may be convenient or desirable in certainapplications (e.g., double-stranded RNAs longer than 25 bp can stimulatethe mammalian immune system, which can be advantageous in certainapplications).

The one or more first caging groups associated with the RNA can becovalently attached to or non-covalently associated with the RNA (e.g.,at one or more bases, riboses, phosphate groups and/or terminalhydroxyls, within and/or at the end of either or both strands of theRNA). In one embodiment, the RNA comprises a first polyribonucleotidecomprising the sense strand and a second polyribonucleotide comprisingthe antisense strand, and the first caging group is covalently attachedto the first polyribonucleotide and to the second polyribonucleotide.For example, the first caging group can be attached to the 5′ end of thefirst polyribonucleotide and to the 3′ end of the secondpolyribonucleotide, or, preferably, it can be attached to the 3′ end ofthe first polyribonucleotide and to the 5′ end of the secondpolyribonucleotide (FIG. 4). The caging group linking the twopolyribonucleotides can, for example, be photolabile.

In a preferred aspect, the one or more first caging groups arephotoactivatable (e.g., photolabile). For example, the caging groups canbe removed by exposure to light with a wavelength between about 60 nmand about 400 nm, between about 400 nm and about 700 nm, and/or betweenabout 700 nm and about 1000 nm. Other caging groups are removable viainput of different uncaging energies; e.g., the one or more caginggroups can be removable by sonication or application of heat, or can beremoved by a chemical or enzyme.

In one class of embodiments, the one or more first caging groups eachcomprises a first binding moiety. The composition also includes a secondbinding moiety that can bind at least one first binding moiety. Forexample, the first binding moiety on the caging groups can comprisebiotin (see, e.g., FIG. 28), and the second binding moiety can compriseavidin or streptavidin. Streptavidin, for example, can thus be bound tothe first caging group, increasing its bulkiness and its effectivenessat inhibiting the caged RNA from participating in RNAi. In someembodiments, the caged RNA comprises two or more first caging groupseach comprising the first binding moiety, and the second binding moietycan bind two or more first binding moieties simultaneously. For example,the caged RNA can comprise at least two biotinylated caging groups(e.g., one at the 5′ end of the sense strand and one at the 5′ end ofthe antisense strand); binding of streptavidin to multiple biotinmoieties on multiple caged RNA molecules links the caged RNAs into alarge network. Cleavage of the photolabile group attaching the biotin tothe RNA results in dissociation of the network. The uncaged RNA can thenparticipate in RNAi.

The RNA optionally also includes at least one label, wherein initiationof RNA interference of the target mRNA by the labeled RNA in the cellresults in an initiation-dependent change in a signal output of thelabel. In a preferred class of embodiments, the label is a fluorescentlabel, and the initiation-dependent change in the signal output of thelabel is a change in fluorescent emission.

In one class of embodiments, the labeled RNA also includes at least onequencher. The label and the quencher are positioned in the RNA such thatfluorescent emission by the label is quenched by the quencher.Initiation of RNA interference by the labeled RNA results in unquenchingof the label (and thus an increase in the fluorescent emission by thelabel). In this class of embodiments, the initiation-dependent change inthe signal output is thus an increase in the fluorescent emission by thelabel. For example, the label and quencher can be positioned on oppositestrands, in close enough proximity to each other that the label isquenched until the sense and antisense strands are separated.

In one class of embodiments in which the RNA comprises a fluorescentlabel and a quencher, the RNA comprises a first polyribonucleotidecomprising the sense strand and a second polyribonucleotide comprisingthe antisense strand. The first polyribonucleotide comprises between 19and 25 nucleotides, the second polyribonucleotide comprises between 19and 25 nucleotides, and the double-stranded region comprises between 19and 25 base pairs. The label and quencher can be attached at essentiallyany suitable positions, e.g., at the 3′ end, at the 5′ end, and/orwithin either or both the first and second polyribonucleotides, e.g., asdescribed for the embodiments above. As noted previously, techniques fordetermining and verifying suitable positions for the label and quencherare well known in the art.

In a related class of embodiments, the labeled RNA comprises twofluorescent labels, one of which is a donor and the other of which is anacceptor. The donor and acceptor are positioned within the RNA such thatenergy transfer (e.g., FRET) occurs between them (e.g., excitation ofthe donor results in fluorescence by the acceptor). Initiation of RNAinterference by the labeled RNA results in loss of energy transferbetween the donor and the acceptor. This can be observed as an increasein fluorescence by the donor or as a decrease in fluorescence by theacceptor. Thus, in a preferred class of embodiments, theinitiation-dependent change in the signal output is a decrease influorescent emission by the acceptor following excitation of the donor.For example, the donor and acceptor can be positioned on oppositestrands, in close enough proximity to each other that energy transferoccurs until the sense and antisense strands are separated.

In one class of embodiments in which the RNA comprises a donor and anacceptor, the RNA comprises a first polyribonucleotide comprising thesense strand and a second polyribonucleotide comprising the antisensestrand. The first polyribonucleotide comprises between 19 and 25nucleotides, the second polyribonucleotide comprises between 19 and 25nucleotides, and the double-stranded region comprises between 19 and 25base pairs. The donor and acceptor can be attached at essentially anysuitable positions, e.g., at the 3′ end, at the 5′ end, and/or withineither or both the first and second polyribonucleotides, e.g., asdescribed for the embodiments above. Techniques for determining andverifying suitable positions for the donor and acceptor are well knownin the art.

In another class of embodiments, the sense strand comprises a firstlabel and the antisense strand a second label. The two labels aredifferent, non-interacting fluorophores with distinct emission spectra(e.g., red and green, such that the double-stranded RNA is yellow whilethe single strands are red and green).

The composition optionally also includes the target mRNA and/or a cell,e.g., a cell comprising the caged RNA and/or the target mRNA. Varioustechniques (e.g., lipofection, microinjection, or electroporation) canbe used to introduce the caged RNA into the cell. In one class ofembodiments, the caged RNA also includes a cellular delivery module,associated with the RNA, that can mediate introduction of the caged RNAinto the cell. All of the various optional configurations and featuresnoted for the embodiments above apply here as well, to the extent theyare relevant, e.g., for types of cellular delivery modules (e.g.,polypeptides, amphipathic peptides, protein transduction domains, andlipids), use of one or more second caging groups, and the like.

Optionally, in the embodiments herein, the caged RNA is bound to amatrix (e.g., electrostatically, covalently, directly or via a linker).In one aspect, the matrix is a surface and the RNA is bound to thesurface at a predetermined location within an array comprising otherRNAs. In other embodiments, the matrix comprises a bead (e.g.,color-coded or otherwise addressable).

Kits for making the caged RNA (e.g., comprising an RNA, one or morefirst caging groups, and instructions for assembling the RNA and thefirst caging groups to form the caged RNA, packaged in one or morecontainers, and/or one or more first caging groups and instructions forassembling the first caging groups and an RNA supplied by a user of thekit to form the caged RNA, packaged in one or more containers) are alsoa feature of the invention. Similarly, the invention provides kits formaking caged and labeled RNA, e.g., a kit comprising one or more firstcaging groups, at least one label, and instructions for assembling thefirst caging groups, at least one label, and an RNA supplied by a userof the kit to form the caged RNA, packaged in one or more containers.

Kits comprising the caged RNA are another feature of the invention. Forexample, one class of embodiments provides a kit comprising the cagedRNA and one or more of: instructions for using the caged RNA (e.g., toattenuate and/or to detect expression of the target mRNA in a cell), adelivery reagent that can mediate introduction of the caged RNA into acell, or a buffer, packaged in one or more containers.

Caging the interfering RNA allows, e.g., precise control over the timingof gene silencing by controlling initiation of RNA interference (alsocalled RNAi or RNA-mediated interference). Use of RNAi for inhibitinggene expression in a number of cell types (including, e.g., mammaliancells) and organisms is well described in the literature, as are methodsfor determining appropriate interfering RNA(s) to target a desired geneand for generating such interfering RNAs. For example, RNA interferenceis described e.g., in U.S. patent application publications 20020173478,20020162126, and 20020182223 and in Hannon G. J. “RNA interference”Nature. Jul. 11, 2002;418 (6894):244-51; Ueda R. “RNAi: a new technologyin the post-genomic sequencing era” J Neurogenet. 2001;15(3-4):193-204;Ullu et al “RNA interference: advances and questions” Philos Trans R SocLond B Biol Sci. Jan. 29, 2002;357(1417):65-70; and Schmid et al“Combinatorial RNAi: a method for evaluating the functions of genefamilies in Drosophila” Trends Neurosci. Feb. 25, 2002 (2):71-4. A kitfor producing interfering RNAs is commercially available, e.g., fromAmbion, Inc. (www.ambion.com, the Silencer™ siRNA construction kit);kits for randomly labeling such RNAs are available from the same source.

As noted, single-stranded siRNAs can also initiate RNAi. Thus, another,related general class of embodiments provides a caged interfering RNA.The caged RNA includes an RNA comprising a single polyribonucleotidestrand comprising an antisense strand, the antisense strand comprising aregion which is substantially complementary to a region of a targetmRNA. The caged RNA also includes one or more first caging groupsassociated with the RNA. The first caging groups inhibit (e.g., prevent)the RNA from initiating RNA interference of the target mRNA in a cellcomprising the caged RNA. The RNA is typically not self-complementary.

The single-stranded RNA can have any of a variety of lengths. Forexample, the polyribonucleotide strand can comprise between 10 and 100nucleotides, between 10 and 80 nucleotides, between 10 and 50nucleotides, preferably between 10 and 30 nucleotides, or morepreferably between 15 and 30 nucleotides or between 19 and 25nucleotides.

The RNA optionally comprises at least one label. In one class ofembodiments, initiation of RNA interference of the target mRNA by thelabeled RNA in the cell results in an initiation-dependent change in asignal output of the label. For example, the single-stranded RNA canhave a fluorescent label at or near one end of the polyribonucleotideand a quencher at or near the other end, or it can have a donor at ornear one end of the polyribonucleotide and an acceptor at or near theother end. Alternatively, signal output of the label can be unaffectedby participation of the RNA in the RNAi pathway.

All of the various optional configurations and features noted for theembodiments above apply here as well, to the extent they are relevant,e.g., for label configurations (e.g., use of fluorescent labels,fluorescent label/quencher, and donor/acceptor combinations), signaloutput types, use of caging groups (e.g., photolabile caging groups),appropriate uncaging energies (light, heat, sonic, etc.), use ofcellular delivery modules (e.g., amphipathic peptides, cationicpeptides, protein transduction domains, and lipids), and the like.

Methods

In one class of methods of the invention, methods of selectivelyattenuating expression of a target gene in a cell are provided. In themethods, a caged RNA is introduced into the cell. The caged RNA caninclude an RNA comprising at least one double-stranded region, thedouble-stranded region comprising a sense strand and an antisensestrand, the antisense strand comprising a region which is substantiallycomplementary to a region of a target mRNA corresponding to the targetgene. Alternatively, the caged RNA can include an RNA comprising asingle polyribonucleotide strand comprising an antisense strand, theantisense strand comprising a region which is substantiallycomplementary to a region of a target mRNA corresponding to the targetgene. The caged RNA comprises one or more caging groups associated withthe RNA, the caging groups inhibiting (e.g., preventing) the RNA frominitiating RNA interference of the target mRNA in the cell. RNAinterference is initiated by exposing the cell to uncaging energy (e.g.,light of a predetermined wavelength), freeing the RNA from inhibition bythe caging groups.

Exposing the cell to uncaging energy optionally includes exposing thecell to light of a first wavelength. This exposure can be addressable;e.g., the caged RNA can be exposed to light of the first wavelength byexposing one or more preselected areas (e.g., wells of a microtiterplate or portions thereof, or the like) to the light. As anotherexample, the uncaging energy can be directed at a preselected subset ofa cell population comprising the cell.

Exposing the cell to light of the first wavelength optionally comprisesexposing the cell to light such that the intensity of the light and theduration of exposure to the light are controlled such that a firstportion (which can be a selected amount) of the caged labeled RNA isuncaged and a second portion of the caged labeled RNA remains caged. Putanother way, the uncaging rate can be controlled. Furthermore, theuncaging step can be repeated until the caged RNA is depleted.

As noted, caging the RNA permits temporal control over initiation of RNAinterference. For example, the method can include contacting the celland a test compound and exposing the cell to the uncaging energy at apreselected time point with respect to a time at which the cell and thetest compound are contacted.

All of the above optional method variations apply to this method aswell. Further, the various composition components noted (particularlythe caged RNA embodiments) above can be adapted for use in this method,as appropriate. For example, in one class of embodiments, the caged RNAfurther comprises a cellular delivery module that can mediateintroduction of the caged RNA into the cell, the cellular deliverymodule being associated with the RNA. In this class of embodiments, thecaged RNA is introduced into the cell by contacting the cell with thecaged RNA associated with the cellular delivery module. As anotherexample, the cellular delivery module can be covalently attached to theRNA via a photolabile linker, which can be cleaved by exposure to lightof an appropriate wavelength once the RNA is inside the cell.

As another example, in one class of embodiments, the RNA comprises atleast one label (e.g., one with an initiation-dependent signal output),and the methods include detecting a signal from the label.

The methods optionally include introducing a plurality of caged RNAsinto the cell. The plurality of caged RNAs can then be uncagedsimultaneously or at different times. For example, a first caged RNA canbe uncaged, e.g., by exposure to light of a first wavelength, andpermitted to initiate RNAi of a first target mRNA. A second caged RNAcan be uncaged, e.g., by exposure to light of a second, differentwavelength, at a later time.

In another aspect, systems and/or apparatus comprising the compositions(e.g., the caged RNAs) noted above and, e.g., components such asdetectors, fluid handling apparatus, sources of uncaging energy, or thelike, are a feature of the invention.

Other Caged RNAs

The presence of RNA, particularly double-stranded RNA, in a cell canresult in inhibition of expression of a gene comprising a sequenceidentical or nearly identical to that of the RNA through mechanismsother than RNAi. For example, double-stranded RNAs that are partiallycomplementary to a target mRNA can repress translation of the mRNAwithout affecting its stability. As another example, double-strandedRNAs can induce histone methylation and heterochromatin formation,leading to transcriptional silencing of a gene comprising a sequenceidentical or nearly identical to that of the RNA (see, e.g., Schramkeand Allshire (2003) “Hairpin RNAs and retrotransposon LTRs effect RNAiand chromatin-based gene silencing” Science 301:1069-1074; Kawasaki andTaira (2004) “Induction of DNA methylation and gene silencing by shortinterfering RNAs in human cells” Nature 431:211-217; and Morris et al.(2004) “Small interfering RNA-induced transcriptional gene silencing inhuman cells” Science 305:1289-1292).

Short RNAs called microRNAs (miRNAs) have been identified in a varietyof species. Typically, these endogenous RNAs are each transcribed as along RNA and then processed to a pre-miRNA of approximately 60-75nucleotides that forms an imperfect hairpin (stem-loop) structure. Thepre-miRNA is typically then cleaved, e.g., by Dicer, to form the maturemiRNA. Mature miRNAs are typically approximately 21-25 nucleotides inlength, but can vary, e.g., from about 14 to about 25 or morenucleotides. Some, though not all, miRNAs have been shown to inhibittranslation of mRNAs bearing partially complementary sequences. SuchmiRNAs contain one or more internal mismatches to the corresponding mRNAthat are predicted to result in a bulge in the center of the duplexformed by the binding of the miRNA antisense strand to the mRNA (e.g.,FIG. 32). The miRNA typically forms approximately 14-17 Watson-Crickbase pairs with the mRNA; additional wobble base pairs can also beformed. In addition, short synthetic double-stranded RNAs (e.g., similarto siRNAs) containing central mismatches to the corresponding mRNA havebeen shown to repress translation (but not initiate degradation) of themRNA. See, for example, Zeng et al. (2003) “MicroRNAs and smallinterfering RNAs can inhibit mRNA expression by similar mechanisms”Proc. Natl. Acad. Sci. USA 100:9779-9784; Doench et al. (2003) “siRNAscan function as miRNAs” Genes & Dev. 17:438-442; Bartel and Bartel(2003) “MicroRNAs: At the root of plant development?” Plant Physiology132:709-717; Schwarz and Zamore (2002) “Why do miRNAs live in themiRNP?” Genes & Dev. 16:1025-1031; Tang et al. (2003) “A biochemicalframework for RNA silencing in plants” Genes & Dev. 17:49-63; Meister etal. (2004) “Sequence-specific inhibition of microRNA- and siRNA-inducedRNA silencing” RNA 10:544-550; Nelson et al. (2003) “The microRNA world:Small is mighty” Trends Biochem. Sci. 28:534-540; Scacheri et al. (2004)“Short interfering RNAs can induce unexpected and divergent changes inthe levels of untargeted proteins in mammalian cells” Proc. Natl. Acad.Sci. USA 101:1892-1897; Sempere et al. (2004) “Expression profiling ofmammalian microRNAs uncovers a subset of brain-expressed microRNAs withpossible roles in murine and human neuronal differentiation” GenomeBiology 5:R13; Dykxhoorn et al. (2003) “Killing the messenger: ShortRNAs that silence gene expression” Nature Reviews Molec. and Cell Biol.4:457-467; McManus (2003) “MicroRNAs and cancer” Semin Cancer Biol.13:253-288; and Stark et al. (2003) “Identification of DrosophilamicroRNA targets” PLoS Biol. 1:E60.

The cellular machinery involved in translational repression of mRNAs bypartially complementary RNAs (e.g., certain miRNAs) appears to partiallyoverlap that involved in RNAi, although, as noted, translation of themRNAs, not their stability, is affected and the mRNAs are typically notdegraded.

The location and/or size of the bulge(s) formed when the antisensestrand of the RNA binds the mRNA can affect the ability of the RNA torepress translation of the mRNA. Similarly, location and/or size of anybulges within the RNA itself can also affect efficiency of translationalrepression. See, e.g., the references above. Typically, translationalrepression is most effective when the antisense strand of the RNA iscomplementary to the 3′ untranslated region (3′ UTR) of the mRNA.Multiple repeats, e.g., tandem repeats, of the sequence complementary tothe antisense strand of the RNA can also provide more effectivetranslational repression; for example, some mRNAs that aretranslationally repressed by endogenous miRNAs contain 7-8 repeats ofthe miRNA binding sequence at their 3′ UTRs. It is worth noting thattranslational repression appears to be more dependent on concentrationof the RNA than RNA interference does; translational repression isthought to involve binding of a single mRNA by each repressing RNA,while RNAi is thought to involve cleavage of multiple copies of the mRNAby a single siRNA-RISC complex.

Guidance for design of a suitable RNA to repress translation of a giventarget mRNA can be found in the literature (e.g., the references aboveand Doench and Sharp (2004) “Specificity of microRNA target selection intranslational repression” Genes & Dev. 18:504-511; Rehmsmeier et al.(2004) “Fast and effective prediction of microRNA/target duplexes” RNA10:1507-1517; Robins et al. (2005) “Incorporating structure to predictmicroRNA targets” Proc Natl Acad Sci 102:4006-4009; and Mattick andMakunin (2005) “Small regulatory RNAs in mammals” Hum. Mol. Genet.14:R121-R132, among many others) and herein. However, due to differencesin efficiency of translational repression between RNAs of differentstructure (e.g., bulge size, sequence, and/or location) and RNAscorresponding to different regions of the target mRNA, several RNAs areoptionally designed and tested against the target mRNA to determinewhich is most effective at repressing translation of the target mRNA(preferably, without inducing endonucleolytic cleavage and degradationof the target mRNA).

The present invention provides a number of novel methods, compositions,systems, and kits related to translational repression and chromatinsilencing by RNAs. In one aspect, caging groups (e.g., photolabilecaging groups) are used to precisely control the timing and/or locationof translational repression or chromatin silencing.

Compositions, Systems, and Kits

One general class of embodiments provides a caged RNA. The caged RNAincludes an RNA capable of repressing translation of a target mRNA. Thecaged RNA also includes one or more first caging groups associated withthe RNA (e.g., two or more, three or more, four or more, or the like,first caging groups). The first caging groups inhibit (e.g., prevent)the RNA from repressing translation of the target mRNA in a cellcomprising the caged RNA.

The first caging groups can inhibit the RNA from repressing translationof the target mRNA by at least about 25%, at least about 30%, at leastabout 35%, at least about 50%, at least about 75%, at least about 90%,at least about 95%, or at least about 98%, as compared to the RNA in theabsence of the first caging groups. In one class of embodiments, thefirst caging groups prevent the RNA from repressing translation of thetarget mRNA (i.e., introduction of the caged RNA into a cell has noeffect on translation of the target mRNA). Removal of or an inducedconformational change in the first caging groups typically permits theRNA to repress translation of the target MRNA.

Note that the first caging groups “inhibiting the RNA from repressingtranslation of the target mRNA” is not meant to imply that the firstcaging groups inhibit any particular step(s) in the translationalrepression pathway. For example, the first caging groups can interferewith cleavage of a long RNA hairpin by Dicer, RISC binding to the RNA,strand separation of the sense and antisense strands of the RNA,antisense strand binding to the target mRNA, and/or any other step in apathway leading to translational repression of the target mRNA.Translational repression can be detected, for example, by a decrease inexpression level of a protein translated from the target mRNA. Methodsfor detecting protein expression levels are well known in the art, andinclude Western analysis, immunoprecipitation, and specific proteinactivity assays, among many others.

In a preferred class of embodiments, the RNA does not initiatedegradation of the target mRNA in a cell comprising the RNA. Thus, forexample, the RNA preferably does not initiate endonucleolytic cleavageand/or RNAi of the target mRNA. Alternatively, the RNA can initiatedegradation of the target mRNA as well as repress its translation; e.g.,expression of the target mRNA can be decreased by at least about 0.1%,1%, 5%, 10%, or even more, while translation of any remaining mRNA isrepressed.

The RNA can have any of a variety of structures, lengths, and/or thelike. For example, the RNA can be single-stranded, or, preferably,double-stranded. The RNA typically comprises at least an antisensestrand (e.g., only the antisense strand if the RNA is single-stranded,or the antisense strand and a complementary or partially complementarysense strand if the RNA is double-stranded). As noted, efficienttranslational repression generally requires at least one mismatchbetween the antisense strand and the target mRNA. Thus, in one class ofembodiments, the antisense strand of the RNA comprises a first regionwhich is complementary to a second region of the target mRNA. The firstregion is interrupted by one or more nucleotides which are notcomplementary to the second region; for example, one or more nucleotideswhich form a bulge when the antisense strand binds the target mRNA. Thefirst region is optionally interrupted by two, three, four, or more(typically at most ten) nucleotides which are not complementary to thesecond region. These nucleotides are typically, but not necessarily,consecutive (e.g., a duplex formed between the antisense strand and thetarget mRNA can have one, two, or more bulges, loops, or the like). Asanother example, the first region can be shorter than the second region,lacking one or more nucleotides corresponding to, e.g., the middle ofthe second region, such that a bulge forms in the mRNA strand of anantisense strand-target mRNA duplex. Similarly, the second region can beshorter than the first region.

The second region, the region of the mRNA to which the antisense strandbinds, can be located essentially anywhere within the mRNA, e.g., the 5′UTR, an exon, an exon, an exon-intron boundary, or the like. In oneclass of embodiments, the second region is within the 3′ UTR of thetarget mRNA. The target optionally includes a plurality of repeats ofthe second region, e.g., tandem repeats, and/or a region complementaryto a different RNA capable of repressing translation of the target.

The RNA optionally comprises at least one double-stranded region thatincludes the antisense strand and a sense strand. The sense strand canbe completely complementary to the antisense strand over thedouble-stranded region. Alternatively, in some embodiments, the sensestrand is not completely complementary to the antisense strand over thedouble-stranded region. For example, the double-stranded region caninclude one or more mismatches, bulges, loops, and/or the like. Themismatched nucleotides can be the same or different nucleotides thanthose mismatched to the target mRNA.

In one class of embodiments, the RNA comprises a firstpolyribonucleotide comprising the sense strand and a secondpolyribonucleotide comprising the antisense strand. For example, thefirst polyribonucleotide can comprise between 14 and 29 nucleotides(e.g., between 17 and 29 or between 18 and 25 nucleotides), the secondpolyribonucleotide can comprise between 14 and 29 nucleotides (e.g.,between 17 and 29 or between 18 and 25 nucleotides), and thedouble-stranded region can comprise between 14 and 29 base pairs (e.g.,between 17 and 29 or between 18 and 25 base pairs). The first and secondpolyribonucleotides can form a duplex over their entire length, or theycan have overhangs. For example, in some embodiments, the firstpolyribonucleotide and the second polyribonucleotide each comprise a twonucleotide TT 3′ overhang (where T is 2′-deoxythymidine). In oneembodiment, at least one of the one or more first caging groups iscovalently attached to a 5′ hydroxyl or a 5′ phosphate of the secondpolyribonucleotide.

As noted, in certain embodiments, the RNA comprises a firstpolyribonucleotide comprising the sense strand and a secondpolyribonucleotide comprising the antisense strand. In otherembodiments, the RNA comprises a self-complementary polyribonucleotide(e.g., a hairpin, a perfect or imperfect hairpin). The stem and/or loopof the hairpin can be any of a variety of different lengths. Forexample, the hairpin can correspond to a pre-miRNA that is processed ina cell to produce an RNA that represses translation of the target mRNA.

The RNA is optionally nuclease resistant and optionally comprises one ormore deoxyribonucleotides, one or more PNA monomers, and/or one or moremodified nucleotides (e.g., 2′-methyl or 2′-O-allyl ribonucleotides) orinternucleotide linkages (e.g., phosphorothioate linkages).

The one or more first caging groups associated with the RNA can becovalently attached to or non-covalently associated with the RNA (e.g.,at one or more bases, riboses, phosphate groups and/or terminalhydroxyls, within and/or at the end of either or both strands of theRNA). In one embodiment, the RNA comprises a first polyribonucleotidecomprising the sense strand and a second polyribonucleotide comprisingthe antisense strand, and the first caging group is covalently attachedto the first polyribonucleotide and to the second polyribonucleotide.For example, the first caging group can be attached to the 5′ end of thefirst polyribonucleotide and to the 3′ end of the secondpolyribonucleotide, or, preferably, it can be attached to the 3′ end ofthe first polyribonucleotide and to the 5′ end of the secondpolyribonucleotide. The caging group linking the two polyribonucleotidescan, for example, be photolabile.

In a preferred aspect, the one or more first caging groups arephotoactivatable (e.g., photolabile). For example, the caging groups canbe removed by exposure to light with a wavelength between about 60 nmand about 400 nm, between about 400 nm and about 700 nm, and/or betweenabout 700 nm and about 1000 nm. Other caging groups are removable viainput of different uncaging energies; e.g., the one or more caginggroups can be removable by sonication or application of heat, or can beremoved by a chemical or enzyme.

In one class of embodiments, the one or more first caging groups eachcomprises a first binding moiety. The composition also includes a secondbinding moiety that can bind at least one first binding moiety. Forexample, the first binding moiety on the caging groups can comprisebiotin, and the second binding moiety can comprise avidin orstreptavidin, as described in the RNAi embodiments above. In someembodiments, the caged RNA comprises two or more first caging groupseach comprising the first binding moiety, and the second binding moietycan bind two or more first binding moieties simultaneously. For example,the caged RNA can comprise at least two biotinylated caging groups(e.g., one at the 5′ end of the sense strand and one at the 5′ end ofthe antisense strand); binding of streptavidin to multiple biotinmoieties on multiple caged RNA molecules links the caged RNAs into alarge network. Cleavage of the photolabile group attaching the biotin tothe RNA results in dissociation of the network. The uncaged RNA can thenparticipate in translational repression.

In some embodiments, the RNA also includes at least one label, e.g., afluorescent label. Optionally, binding and/or repression of translationof the target mRNA by the RNA results in a binding and/orrepression-dependent change in a signal output of the label. The labeledRNA optionally also includes at least one quencher. For example, thelabel and quencher can be positioned on opposite strands of the RNA, inclose enough proximity to each other that the label is quenched untilthe sense and antisense strands are separated. In a related class ofembodiments, the labeled RNA comprises two fluorescent labels, one ofwhich is a donor and the other of which is an acceptor. The donor andacceptor are positioned within the RNA such that energy transfer (e.g.,FRET) occurs between them (e.g., excitation of the donor results influorescence by the acceptor). For example, the donor and acceptor canbe positioned on opposite strands, in close enough proximity to eachother that energy transfer occurs until the sense and antisense strandsare separated. In embodiments in which the RNA includes a sense strandand the antisense strand, the sense strand can comprise a first labeland the antisense strand a second label. The two labels can bedifferent, non-interacting fluorophores with distinct emission spectra(e.g., red and green, such that the double-stranded RNA is yellow whilethe single strands are red and green). As noted previously, techniquesfor determining and verifying suitable positions for the label(s) orlabel and quencher are well known in the art.

The composition optionally also includes the target mRNA and/or a cell,e.g., a cell comprising the caged RNA and/or the target mRNA. Varioustechniques (e.g., lipofection, microinjection, or electroporation) canbe used to introduce the caged RNA into the cell. In one class ofembodiments, the caged RNA also includes a cellular delivery module,associated with the RNA, that can mediate introduction of the caged RNAinto the cell. All of the various optional configurations and featuresnoted for the embodiments above apply here as well, to the extent theyare relevant, e.g., for types of cellular delivery modules (e.g.,polypeptides, amphipathic peptides, protein transduction domains, andlipids), use of the first caging group as a cellular delivery module,use of one or more second caging groups, and the like.

Optionally, in the embodiments herein, the caged RNA is bound to amatrix (e.g., electrostatically, covalently, directly or via a linker).In one aspect, the matrix is a surface and the RNA is bound to thesurface at a predetermined location within an array comprising otherRNAs. In other embodiments, the matrix comprises a bead (e.g.,color-coded or otherwise addressable).

Kits for making the caged RNA are also a feature of the invention. Thus,one class of embodiments provides a kit including an RNA, one or morefirst caging groups, and instructions for assembling the RNA and thefirst caging groups to form the caged RNA, packaged in one or morecontainers. Another class of embodiments provides a kit comprising oneor more first caging groups and instructions for assembling the firstcaging groups and an RNA supplied by a user of the kit to form the cagedRNA, packaged in one or more containers.

Kits comprising the caged RNA are another feature of the invention. Forexample, one class of embodiments provides a kit comprising the cagedRNA and one or more of: instructions for using the caged RNA (e.g., toattenuate expression of the target mRNA in a cell), a delivery reagentthat can mediate introduction of the caged RNA into a cell, or a buffer,packaged in one or more containers.

All of the various optional configurations and features noted for theembodiments above apply here as well, to the extent they are relevant,e.g., for label configurations (e.g., use of fluorescent labels,fluorescent label/quencher, and donor/acceptor combinations), signaloutput types, appropriate uncaging energies (light, heat, sonic, etc.),and the like.

In another aspect, systems and/or apparatus comprising the compositions(e.g., the caged RNAs) noted above and, e.g., components such asdetectors, fluid handling apparatus, sources of uncaging energy, or thelike, are a feature of the invention.

Another aspect of the invention deals with RNAs capable of inducinghistone methylation and chromatin silencing. Such RNAs can be designedand tested by techniques known in the art, e.g., for assayingheterochromatin formation, mRNA expression levels, and the like.

Thus, one general class of embodiments provides a caged RNA thatincludes an RNA capable of silencing transcription of a target gene andone or more first caging groups associated with the RNA. The firstcaging groups inhibit (e.g., prevent) the RNA from silencingtranscription of the target gene in a cell comprising the caged RNA.Transcriptional silencing can reduce the amount of target mRNA presentin a cell; for example, expression of the target mRNA can be decreasedby at least about 5%, at least about 10%, at least about 25%, at leastabout 50%, or at least about 75% or more, or can even be reduced to anundetectable level.

All of the various optional configurations and features noted for theembodiments above apply here as well, to the extent they are relevant,e.g., for percent inhibition by the caging groups, structure of the RNA,label configurations (e.g., use of fluorescent labels, fluorescentlabel/quencher, and donor/acceptor combinations), signal output types,use of caging groups (e.g., photolabile caging groups), appropriateuncaging energies (light, heat, sonic, etc.), use of cellular deliverymodules (e.g., amphipathic peptides, cationic peptides, proteintransduction domains, and lipids), and the like.

In another aspect, systems and/or apparatus comprising the compositions(e.g., the caged RNAs) noted above and, e.g., components such asdetectors, fluid handling apparatus, sources of uncaging energy, or thelike, are a feature of the invention, as are kits for making or usingthe caged RNAs.

Methods

In one class of methods of the invention, methods of selectivelyattenuating expression of a target gene in a cell are provided. In themethods, a caged RNA is introduced into the cell. The caged RNA includesan RNA capable of repressing translation of a target mRNA transcribedfrom the target gene. The caged RNA also comprises one or more caginggroups associated with the RNA, the caging groups inhibiting (e.g.,preventing) the RNA from repressing translation of the target mRNA inthe cell. Repression of translation is initiated by exposing the cell touncaging energy (e.g., light of a predetermined wavelength), freeing theRNA from inhibition by the caging groups. In a preferred class ofembodiments, the amount of the target mRNA present in the cell is notaffected by the presence of the RNA in the cell; i.e., uncaging the RNAdoes not initiate RNAi.

Exposing the cell to uncaging energy optionally includes exposing thecell to light of a first wavelength. This exposure can be addressable;e.g., the caged RNA can be exposed to light of the first wavelength byexposing one or more preselected areas (e.g., wells of a microtiterplate or portions thereof, or the like) to the light. As anotherexample, the uncaging energy can be directed at a preselected subset ofa cell population comprising the cell.

Exposing the cell to light of the first wavelength optionally comprisesexposing the cell to light such that the intensity of the light and theduration of exposure to the light are controlled such that a firstportion (which can be a selected amount) of the caged RNA is uncaged anda second portion of the caged RNA remains caged. Put another way, theuncaging rate can be controlled. Furthermore, the uncaging step can berepeated until the caged RNA is depleted.

As noted, caging the RNA permits temporal control over initiation oftranslational repression. For example, the method can include contactingthe cell and a test compound and exposing the cell to the uncagingenergy at a preselected time point with respect to a time at which thecell and the test compound are contacted.

All of the above optional method variations apply to this method aswell. Further, the various composition components noted (particularlythe caged RNA embodiments) above can be adapted for use in this method,as appropriate. For example, in one class of embodiments, the caged RNAfurther comprises a cellular delivery module that can mediateintroduction of the caged RNA into the cell, the cellular deliverymodule being associated with the RNA. In this class of embodiments, thecaged RNA is introduced into the cell by contacting the cell with thecaged RNA associated with the cellular delivery module. As anotherexample, the cellular delivery module can be covalently attached to theRNA via a photolabile linker, which can be cleaved by exposure to lightof an appropriate wavelength once the RNA is inside the cell.

As another example, in one class of embodiments, the RNA comprises atleast one label (e.g., one with a binding and/or repression-dependentsignal output), and the methods include detecting a signal from thelabel.

The methods optionally include introducing a plurality of caged RNAsinto the cell. The plurality of caged RNAs can then be uncagedsimultaneously or at different times. For example, a first caged RNA canbe uncaged, e.g., by exposure to light of a first wavelength, andpermitted to repress translation of a first target mRNA. A second cagedRNA can be uncaged, e.g., by exposure to light of a second, differentwavelength, at a later time.

Another class of methods of the invention also provides methods ofselectively attenuating expression of a target gene in a cell. In themethods, a caged RNA is introduced into the cell. The caged RNAcomprises an RNA capable of silencing transcription of the target gene.The caged RNA also includes one or more first caging groups associatedwith the RNA that inhibit (e.g., prevent) the RNA from silencingtranscription of the target gene in the cell. Silencing of transcriptionof the target gene is initiated by exposing the cell to uncaging energy,freeing the RNA from inhibition by the caging groups.

All of the above optional method variations apply to this method aswell, e.g., for types of uncaging energy, temporal and spatial controlof uncaging, introduction of the RNA into the cell through use of acellular delivery module, label detection, and the like.

Interfering RNAs with Protein Transduction Domains

As noted, interfering RNAs and other RNAs can be introduced into cellsusing protein transduction domains. Thus, one class of embodimentsprovides a composition comprising an RNA and a protein transductiondomain covalently attached to the RNA. The RNA can comprise at least onedouble-stranded region, the double-stranded region comprising a sensestrand and an antisense strand, the antisense strand comprising a regionwhich is substantially complementary to a region of a target mRNA.Alternatively, the RNA can comprise a single polyribonucleotide strandcomprising an antisense strand, the antisense strand comprising a regionwhich is substantially complementary to a region of a target mRNAcorresponding to the target gene. The composition optionally alsoincludes the target mRNA and/or a cell, e.g., a cell comprising the RNAand/or the target mRNA.

The RNA can be, for example, an interfering RNA, an RNA capable ofrepressing translation of the target mRNA, or an RNA capable ofsilencing transcription of a gene from which the target mRNA istranscribed. Thus, in some embodiments, the region of the antisensestrand that is substantially complementary to a region of the targetmRNA is completely complementary to the region of the target mRNA. Inother embodiments, the region of complementarity is interrupted. Forexample, the region of the antisense strand that is substantiallycomplementary to the region of the target mRNA can comprise at least afirst and a second subregion, each of which is completely complementaryto the target mRNA, flanking one or more nucleotides (e.g., two, three,four, or more nucleotides) which are not complementary to the targetmRNA.

The protein transduction domain can be essentially any proteintransduction domain that can mediate introduction of the RNA into thecell. In one class of embodiments, the protein transduction domain isderived from an HIV-1 Tat protein, from a herpes simplex virus VP22protein, or from a Drosophila antennapedia protein (e.g., Penetratin™).In other embodiments, the protein transduction domain is a model proteintransduction domain, e.g., a homopolymer of lysine, histidine, orD-arginine, e.g., 8-D-Arg.

The covalent attachment between the protein transduction domain and theRNA is optionally reversible by exposure to light of a preselectedwavelength. Similarly, the protein transduction domain can be attachedto the RNA through a disulfide bond or an ester linkage that can bereduced or cleaved once the RNA is inside the cell.

All of the various optional configurations and features noted for theembodiments above apply here as well, to the extent they are relevant,e.g., for RNA configurations (e.g., one or two polyribonucleotides, ofvarious lengths, with or without overhangs, etc.), use of caging groups(e.g., photolabile caging groups), appropriate uncaging energies (light,heat, sonic, etc.), label configurations (e.g., use of fluorescentlabels, fluorescent label/quencher, and donor/acceptor combinations),signal output types, binding to a matrix, and the like.

Kits for making the protein transduction domain-linked RNAs are also afeature of the invention. For example, one embodiment provides a kitcomprising an RNA, a protein transduction domain, and instructions forassembling the RNA and the protein transduction domain to form thecomposition, packaged in one or more containers. A related embodimentprovides a kit comprising a protein transduction domain and instructionsfor assembling the protein transduction domain and an RNA supplied by auser of the kit to form the composition, packaged in one or morecontainers.

The invention also provides related methods of introducing an RNA into acell. In the methods, a composition comprising an RNA and a proteintransduction domain covalently attached to the RNA is provided. The RNAcan comprise at least one double-stranded region, the double-strandedregion comprising a sense strand and an antisense strand, the antisensestrand comprising a region which is substantially complementary to aregion of a target mRNA. Alternatively, the RNA can comprise a singlepolyribonucleotide strand comprising an antisense strand, the antisensestrand comprising a region which is substantially complementary to aregion of a target mRNA corresponding to the target gene. Thecomposition and the cell are contacted, whereby the protein transductiondomain mediates introduction of the RNA into the cell.

In some embodiments, the composition comprises one or more first caginggroups associated with the RNA, which inhibit the RNA from initiatingRNA interference of the target mRNA in the cell. The method includesinitiating RNA interference of the target mRNA by exposing the cell touncaging energy of a first type, freeing the RNA from inhibition by thefirst caging groups. In related embodiments, the composition includesone or more first caging groups associated with the RNA, which inhibitthe RNA from repressing translation of the target mRNA in the cell. Themethod then includes initiating translational repression of the targetmRNA by exposing the cell to uncaging energy of a first type, freeingthe RNA from inhibition by the first caging groups. Similarly, thecomposition optionally includes one or more first caging groupsassociated with the RNA, which inhibit the RNA from silencingtranscription of a gene corresponding to the target mRNA in the cell.The method then includes initiating transcriptional silencing of thetarget gene by exposing the cell to uncaging energy of a first type,freeing the RNA from inhibition by the first caging groups.

All of the above optional method variations apply to this method aswell. Further, the various composition components noted (particularlythe protein transduction domain-linked RNA embodiments) above can beadapted for use in this method, as appropriate.

In another aspect, systems and/or apparatus comprising the compositionsnoted above and, e.g., components such as detectors, fluid handlingapparatus, sources of uncaging energy, or the like, are a feature of theinvention.

Interfering RNAs with Lipids

Interfering RNAs and other RNAs can also be introduced into cells bycovalently or non-covalently associated lipids. Thus, one class ofembodiments provides a composition comprising an RNA and a lipidcovalently or non-covalently attached to the RNA. The RNA can compriseat least one double-stranded region, the double-stranded regioncomprising a sense strand and an antisense strand, the antisense strandcomprising a region which is substantially complementary to a region ofa target mRNA; alternatively, the RNA can comprise a singlepolyribonucleotide strand comprising an antisense strand, the antisensestrand comprising a region which is substantially complementary to aregion of a target mRNA. The lipid can be, e.g., a fatty acid. In oneexample class of embodiments, the lipid comprises (or, e.g., consistsof) a myristoyl group.

All of the various optional configurations and features noted for theembodiments above apply here as well, to the extent they are relevant,e.g., for label configurations (e.g., use of fluorescent labels,fluorescent label/quencher, and donor/acceptor combinations), signaloutput types, RNA configurations (e.g., one or two polyribonucleotides,of various lengths, with or without overhangs, etc.), use of caginggroups (e.g., photolabile caging groups), appropriate uncaging energies(light, heat, sonic, etc.), use of cellular delivery modules (e.g.,amphipathic peptides, protein transduction domains, and lipids), and thelike. It is worth noting that the composition optionally also includesthe target mRNA and/or a cell, e.g., a cell comprising the target mRNAand/or the RNA. It is also worth noting that the RNA can be, forexample, an interfering RNA, an RNA capable of repressing translation ofthe target mRNA, or an RNA capable of silencing transcription of a genefrom which the target mRNA is transcribed. Thus, for example, the regionof the antisense strand that is substantially complementary to a regionof the target mRNA can be completely complementary to the region of thetarget mRNA, or the region of complementarity can be interrupted.

Kits for making the lipid-linked RNAs are also a feature of theinvention. For example, one embodiment provides a kit comprising an RNA,a lipid, and instructions for assembling the RNA and the lipid to formthe composition, packaged in one or more containers. A relatedembodiment provides a kit comprising a lipid and instructions forassembling the lipid and an RNA supplied by a user of the kit to formthe composition, packaged in one or more containers.

The invention also provides related methods of introducing an RNA into acell. In the methods, a composition comprising an RNA and a lipidcovalently attached to the RNA is provided. The RNA can comprise atleast one double-stranded region, the double-stranded region comprisinga sense strand and an antisense strand, the antisense strand comprisinga region which is substantially complementary to a region of a targetmRNA; alternatively, the RNA can comprise a single polyribonucleotidestrand comprising an antisense strand, the antisense strand comprising aregion which is substantially complementary to a region of a targetmRNA. The composition and the cell are contacted, whereby the lipidmediates introduction of the RNA into the cell.

All of the above optional method variations apply to this method aswell. Further, the various composition components noted (particularlythe lipid-linked RNA embodiments) above can be adapted for use in thismethod, as appropriate.

In some embodiments, the composition comprises one or more first caginggroups associated with the RNA, which inhibit the RNA from initiatingRNA interference of the target mRNA in the cell. The method includesinitiating RNA interference of the target mRNA by exposing the cell touncaging energy of a first type, freeing the RNA from inhibition by thefirst caging groups. In related embodiments, the composition includesone or more first caging groups associated with the RNA, which inhibitthe RNA from repressing translation of the target mRNA in the cell. Themethod then includes initiating translational repression of the targetmRNA by exposing the cell to uncaging energy of a first type, freeingthe RNA from inhibition by the first caging groups. Similarly, thecomposition optionally includes one or more first caging groupsassociated with the RNA, which inhibit the RNA from silencingtranscription of a gene corresponding to the target mRNA in the cell.The method then includes initiating transcriptional silencing of thetarget gene by exposing the cell to uncaging energy of a first type,freeing the RNA from inhibition by the first caging groups.

In another aspect, systems and/or apparatus comprising the compositionsnoted above and, e.g., components such as detectors, fluid handlingapparatus, sources of uncaging energy, or the like, are a feature of theinvention.

Induction of RNA Expression

In one aspect, the invention includes methods of selectively attenuatingexpression of a target mRNA in a cell. In the methods, one or morevectors that comprise or encode an RNA are introduced into the cell. TheRNA comprises at least one double-stranded region, the double-strandedregion comprising a sense strand and an antisense strand, the antisensestrand comprising a region which is substantially complementary to aregion of the target mRNA. A caged first activation component is alsointroduced into the cell. The caged first activation component includesone or more caging groups associated with a first activation component.The first activation component directly or indirectly increasesexpression of the RNA from the one or more vectors, and the one or morecaging groups inhibit (e.g., prevent) the first activation componentfrom increasing expression of the RNA. The cell is exposed to uncagingenergy (e.g., light of a first wavelength), whereby exposure to theuncaging energy frees the first activation component from inhibition bythe caging groups. This results in increased expression of the RNA,which can then initiate RNA interference of the target mRNA, represstranslation of the target mRNA, or silence transcription of a gene fromwhich the target mRNA is transcribed, for example.

In one class of embodiments, the first activation component directlyincreases expression of the RNA from the one or more vectors. Forexample, the first activation component can be a transcription factor(i.e., a transcriptional activator) or an RNA polymerase, e.g., T7polymerase.

In another class of embodiments, the first activation componentindirectly increases expression of the RNA by binding to a secondactivation component, whereby the bound second activation componentdirectly increases expression of the RNA. An example embodiment isschematically illustrated in FIG. 30, which depicts tetracycline cagedwith a photolabile caging group (the caged first activation component).Exposure to light frees the tetracycline from the caging group. In thisexample, the tetracycline binds a tetracycline-controlled transactivator(tTA, the second activation component), which stimulates transcriptionof the interfering RNA from a promoter comprising tet operatorsequences.

In yet another class of embodiments, the first activation componentindirectly increases expression of the RNA by indirectly activating athird activation component, whereby the activated third activationcomponent directly increases expression of the RNA. An exampleembodiment is schematically illustrated in FIG. 31, which depicts IP3(inositol 1,4,5-triphosphate) caged with a photolabile caging group (thecaged first activation component). Exposure to light frees the IP3 fromthe caging group, leading to a rise in intracellular Ca²⁺ concentration.The increased Ca²⁺ concentration stimulates calcineurin todephosphorylate the NF-AT (nuclear factor of activated T cells)transcription factor complex, which then migrates into the nucleus andactivates expression of the interfering RNA from a promoter comprisingNF-AT-response elements. Caged Ca²⁺, for example, can also be used as afirst activation component in this system.

Other examples of suitable first activation components include, but arenot limited to, cAMP, non-mammalian steroid hormones and small moleculesthat bind immunophilins. See, e.g., Gossen and Bujard (1992) “Tightcontrol of gene expression in mammalian cells by tetracycline-responsivepromoters” Proc. Natl. Acad. Sci. USA 89:5547-5551; Saez et al. (1997)“Inducible gene expression in mammalian cells and transgenic mice” Curr.Opin. Biotechnol. 8:608-616; Li et al. (1998) “Cell-permeant caged InsP3ester shows that Ca2+ spike frequency can optimize gene expression”Nature 392:936-541; and Lin et al. (2002) “Spatially discrete,light-driven protein expression” Chem. Biol. 9:1347-1353.

Methods of expressing interfering RNAs of various lengths and structuresfrom vectors are well known in the art. See, e.g., Patterson and Hannon(2002) “Stable suppression of gene expression by RNAi in mammaliancells” Proc. Natl. Acad. Sci. USA 99:1443-1448 and Garbarek and Glover(2003) “RNA interference by production of short hairpin dsRNA in EScells, their differentiated derivatives, and somatic cell lines”BioTechniques 34:734-744. Methods of expressing other RNAs are similarlyknown.

The invention also provides compositions related to the methods. Thus,one general class of embodiments provides a composition comprising oneor more vectors and a caged first activation component. The one or morevectors comprise or encode an RNA comprising at least onedouble-stranded region, the double-stranded region comprising a sensestrand and an antisense strand, the antisense strand comprising a regionwhich is substantially complementary to a region of a target mRNA. Thecaged first activation component comprises one or more caging groupsassociated with a first activation component, which first activationcomponent directly or indirectly increases expression of the RNA fromthe one or more vectors in a cell comprising the one or more vectors andthe first activation component, and which one or more caging groupsinhibit the first activation component from increasing expression of theRNA in the cell. The composition optionally includes the target mRNAand/or a cell, e.g., a cell comprising the one or more vectors and thecaged first activation component and/or the target mRNA.

The composition optionally also includes a second activation component,which second activation component directly increases expression of theRNA when bound by the first activation component (e.g., tetracycline).In a related class of embodiments, the composition optionally alsoincludes a third activation component, which third activation componentdirectly increases expression of the RNA when indirectly activated bythe first activation component. For example, the first activationcomponent can comprise IP3 or Ca²⁺ and the third activation componentcan comprise an NF-AT transcription factor complex. Other examples ofsuitable first activation components include, but are not limited to,cAMP, non-mammalian steroid hormones and small molecules that bindimmunophilins.

The length and/or structure of the RNA can vary. For example, the RNAcan comprise a first polyribonucleotide comprising the sense strand anda second polyribonucleotide comprising the antisense strand. Thedouble-stranded region formed by annealing of the sense and antisensestrands can, e.g., comprise more than about 1500 base pairs, comprisefewer than about 1500 base pairs, fewer than about 1000 base pairs,fewer than about 500 base pairs, fewer than about 250 base pairs, fewerthan about 150 base pairs, fewer than about 80 base pairs, fewer thanabout 50 base pairs, fewer than about 30 base pairs, or even fewer thanabout 25 base pairs. Instead of comprising a two-stranded interferingRNA (e.g., a siRNA), the RNA comprises a self-complementarypolyribonucleotide (e.g., an shRNA). As noted for the embodiments above,the RNA can be, for example, an interfering RNA, an RNA capable ofrepressing translation of the target mRNA, or an RNA capable ofsilencing transcription of a gene from which the target mRNA istranscribed, for example.

Kits form another feature of the invention. Thus, one class ofembodiments provides a kit comprising one or more vectors and a cagedfirst activation component, packaged in one or more containers. The kitcan also include a vector that comprises or encodes a second or a thirdactivation component, and/or instructions for using the kit, e.g.,instructions for using the kit to attenuate expression of a target mRNA.

All of the various optional configurations and features noted for theembodiments above apply to the methods and compositions here as well, tothe extent they are relevant, e.g., RNA configurations (e.g., one or twopolyribonucleotides, of various lengths, with or without overhangs,etc.), use of caging groups (e.g., photolabile caging groups),appropriate uncaging energies (light, heat, sonic, etc.), use ofcellular delivery modules (e.g., amphipathic peptides, proteintransduction domains, and lipids), and the like.

Caged DNAs Encoding Interfering RNAs and Other RNAs

The invention also includes other methods of selectively attenuatingexpression of a target gene in a cell. In one general class of methods,a first caged DNA and a second caged DNA are introduced into the cell.The first caged DNA includes a first DNA encoding an RNA sense strandand one or more caging groups. The second caged DNA comprises a secondDNA encoding an RNA antisense strand and one or more caging groups. Thepresence of the caging groups prevents transcription of the first andsecond DNAs, the first and second DNAs each comprising at least aportion of the target gene, and the sense and antisense strands being atleast partially complementary and able to form a duplex over at least aportion of their lengths. RNA interference is initiated by generatingdouble-stranded RNA by exposing the cell to uncaging energy, wherebyexposure to the uncaging energy frees the first and second DNAs from thecaging groups and permits transcription of the first and second DNAs tooccur.

The resulting double-stranded RNA can comprise two distinctpolyribonucleotides (i.e., the sense strand can comprise a firstpolyribonucleotide while the antisense strand comprises a secondpolyribonucleotide), or the resulting double-stranded RNA can comprise asingle, self-complementary polyribonucleotide that includes the senseand antisense strands (e.g., an shRNA).

All of the above optional method variations apply to this method aswell, to the extent they are relevant. Further, the various compositioncomponents noted above can be adapted for use in this method, asappropriate; e.g., use of caging groups (e.g., photolabile caginggroups), appropriate uncaging energies (light, heat, sonic, etc.), useof cellular delivery modules (e.g., amphipathic peptides, proteintransduction domains, and lipids), and the like. It is worth noting thatwhen the resulting double-stranded RNA comprises a single,self-complementary polyribonucleotide, the first and second DNAs arecovalently joined in proximity to each other as a single transcriptionunit, e.g., on a plasmid. When the resulting double-stranded RNAcomprises two distinct polyribonucleotides, the first and second DNAscan be on separate plasmids or can optionally be included on a singleplasmid (see, e.g., U.S. patent application publication 20020182223).The DNAs are optionally nuclease resistant.

In another general class of methods, a first caged DNA and a secondcaged DNA are introduced into the cell. The first caged DNA includes afirst DNA encoding an RNA sense strand and one or more caging groups.The second caged DNA comprises a second DNA encoding an RNA antisensestrand and one or more caging groups. The presence of the caging groupsprevents transcription of the first and second DNAs, the first andsecond DNAs each comprising at least a portion of the target gene, andthe sense and antisense strands being at least partially complementaryand able to form a duplex over at least a portion of their lengths.Translational repression is initiated by generating double-stranded RNAby exposing the cell to uncaging energy, whereby exposure to theuncaging energy frees the first and second DNAs from the caging groupsand permits transcription of the first and second DNAs to occur.

The resulting double-stranded RNA can comprise two distinctpolyribonucleotides (i.e., the sense strand can comprise a firstpolyribonucleotide while the antisense strand comprises a secondpolyribonucleotide), or the resulting double-stranded RNA can comprise asingle, self-complementary polyribonucleotide that includes the senseand antisense strands.

All of the above optional method variations apply to this method aswell, to the extent they are relevant. Further, the various compositioncomponents noted above can be adapted for use in this method, asappropriate; e.g., use of caging groups (e.g., photolabile caginggroups), appropriate uncaging energies (light, heat, sonic, etc.), useof cellular delivery modules (e.g., amphipathic peptides, proteintransduction domains, and lipids), and the like. It is worth noting thatwhen the resulting double-stranded RNA comprises a single,self-complementary polyribonucleotide, the first and second DNAs arecovalently joined in proximity to each other as a single transcriptionunit, e.g., on a plasmid. When the resulting double-stranded RNAcomprises two distinct polyribonucleotides, the first and second DNAscan be on separate plasmids or can optionally be included on a singleplasmid, as described above. The DNAs are optionally nuclease resistant.

RNAs capable of silencing transcription of a target gene can besimilarly expressed.

Caging Groups

A large number of caging groups, and a number of reactive compounds thatcan be used to covalently attach caging groups to other molecules, arewell known in the art. Examples of photolabile caging groups include,but are not limited to: 2-nitrobenzyl;1-(4,5-dimethoxy-2-nitrophenyl)ethyl (DMNPE); brominated7-hydroxycoumarin-4-ylmethyls (e.g.,6-Bromo-7-hydroxycoumarin-4-ylmethyl (Bhc)); nitroindolines;N-acyl-7-nitroindolines; phenacyls; hydroxyphenacyl; benzoin esters;dimethoxybenzoin; meta-phenols; 4,5-dimethoxy-2-nitrobenzyl (DMNB);alpha-carboxy-2-nitrobenzyl (CNB); 1-(2-nitrophenyl)ethyl (NPE);5-carboxymethoxy-2-nitrobenzyl (CMNB);(5-carboxymethoxy-2-nitrobenzyl)oxy)carbonyl;(4,5-dimethoxy-2-nitrobenzyl)oxy) carbonyl; desoxybenzoinyl; and thelike. See e.g., WO 2004/046339, U.S. Pat. No. 5,635,608 to Haugland andGee (Jun. 3, 1997) entitled “α-carboxy caged compounds”; Neuro 19, 465(1997); J Physiol 508.3, 801 (1998); Proc Natl Acad Sci USA September1998; 85(17):6571-5; J Biol Chem Feb. 14, 1997; 272(7):4172-8; Neuron20,619-624, 1998; Nature Genetics, vol. 28:2001:317-325; Nature, vol.392,1998:936-941; Pan, P., and Bayley, H. “Caged cysteine andthiophosphoryl peptides” FEBS Letters 405:81-85 (1997); Pettit et al.(1997) “Chemical two-photon uncaging: a novel approach to mappingglutamate receptors” Neuron 19:465-471; Furuta et al. (1999) “Brominated7-hydroxycoumarin-4-ylmethyls: novel photolabile protecting groups withbiologically useful cross-sections for two photon photolysis” Proc.Natl. Acad. Sci. 96(4):1193-1200; Zou et al. “Catalytic subunit ofprotein kinase A caged at the activating phosphothreonine” J. Amer.Chem. Soc. (2002) 124: 8220-8229; Zou et al. “Caged ThiophosphotyrosinePeptides” Angew. Chem. Int. Ed. (2001) 40: 3049-3051; Conrad II et al.“p-Hydroxyphenacyl Phototriggers: The Reactive Excited State ofPhosphate Photorelease” J. Am. Chem. Soc. (2000) 122:9346-9347; ConradII et al. “New Phototriggers 10: Extending the π,π* Absorption toRelease Peptides in Biological Media” Org. Lett. (2000) 2:1545-1547;Givens et al. “A New Phototriggers 9: p-Hydroxyphenacyl as a C-TerminusPhotoremovable Protecting Group for Oligopeptides” J. Am. Chem. Soc.(2000) 122:2687-2697; Bishop et al.“40-Aminomethyl-2,20-bipyridyl-4-carboxylic Acid (Abc) and RelatedDerivatives: Novel Bipyridine Amino Acids for the Solid-PhaseIncorporation of a Metal Coordination Site Within a Peptide Backbone”Tetrahedron (2000)56:4629-4638; Ching et al “Polymers As Surface-BasedTethers with Photolytic triggers Enabling Laser-InducedRelease/Desorption of Covalently Bound Molecules” Bioconjugate Chemistry(1996) 7:525-8; U.S. Pat. No. 5,888,829 to Gee and Millard (Mar. 30,1999) entitled “Photolabile caged ionophores and method of using in amembrane separation process”; U.S. Pat. No. 6,043,065 to Kao et al.(Mar. 28, 2000) entitled “Photosensitive organic compounds that release2,5,-di(tert-butyl) hydroquinone upon illumination”; U.S. Pat. No.5,430,175 to Hess et al. (Jul. 4, 1995) entitled “Caged carboxylcompounds and use thereof”; U.S. Pat. No. 5,872,243; PNAS (1980)77:7237-41; BioProbes Handbook, 2002 from Molecular Probes, Inc.; andHandbook of Fluorescent Probes and Research Products, Ninth Edition orWeb Edition, from Molecular Probes, Inc, as well as the referencesbelow. Many compounds, kits, etc. for use in caging various moleculesare commercially available, e.g., from Molecular Probes, Inc.(www.molecularprobes.com).

Environmentally responsive polymers suitable for use as caging groupshave also been described. Such polymers undergo conformational changesinduced by light, an electric or magnetic field, a change in pH and/orionic strength, temperature, or addition of an antigen or saccharide, orother environmental variables. For example, Shimoboji et al. (2002)“Photoresponsive polymer-enzyme switches” Proc. Natl. Acad. Sci. USA99:16,592-16,596 describes polymers that undergo reversibleconformational changes in response to light; such polymers can, e.g., beused as photoactivatable caging groups. See also Ding et al. (2001)“Size-dependent control of the binding of biotinylated proteins tostreptavidin using a polymer shield” Nature 411:59-62; Miyata et al.(1999) “A reversibly antigen-responsive hydrogel” Nature 399:766-769;Murthy et al. (2003) “Bioinspired pH-responsive polymers for theintracellular delivery of biomolecular drugs” Bioconjugate Chem.14:412-419; and Galaev and Mattiasson (1999) “‘Smart’ polymers and whatthey could do in biotechnology and medicine” Trends Biotech. 17:335-340.FIGS. 26 and 27 schematically illustrate use of environmentallyresponsive polymers as caging groups. FIG. 26 illustrates noncovalentassociation of a polymer with a component to be caged (e.g., an siRNA).In its folded conformation, the polymer physically surrounds and trapsthe component (Panel B). The caged RNA is optionally introduced into acell. A conformational change in the polymer induced by light, pH,temperature, or the like results in release of the RNA from the unfoldedconformation of the polymer (Panel D). FIG. 27 illustrates covalentassociation of a polymer with an example double-stranded siRNA. In itsfolded conformation, the polymer prevents the siRNA from initiating RNAi(e.g., by preventing the siRNA from interacting with a kinase, RISC, orother components of the RNAi cellular machinery) (Panel A). Aconformational change in the polymer induced by light, pH, temperature,or the like permits the siRNA to initiate RNAi (Panel B).

Caged polypeptides (including, e.g., polypeptide cellular deliverymodules, e.g., protein transduction domains) can be produced, forexample, by reacting a polypeptide with a caging compound or byincorporating a caged amino acid during synthesis of a polypeptide. See,e.g., U.S. Pat. No. 5,998,580 to Fay et al. (Dec. 7, 1999) entitled“Photosensitive caged macromolecules”; Kossel et al. (2001) PNAS98:14702-14707; Trends Plant Sci (1999) 4:330-334; PNAS (1998)95:1568-1573; J Am Chem Soc (2002) 124:8220-8229; Pharmacology &Therapeutics (2001) 91:85-92; and Angew Chem Int Ed Engl (2001)40:3049-3051. A photolabile polypeptide linker (e.g., for connecting aprotein transduction domain and an RNA, or the like) can, for example,comprise a photolabile amino acid such as that described in U.S. Pat.No. 5,998,580 (supra).

Caged nucleic acids (e.g., DNA, RNA or PNA, e.g., interfering RNAs) canbe produced by reacting the nucleic acids with caging compounds or byincorporating a caged nucleotide during synthesis of a nucleic acid. Forexample, U.S. Pat. No. 6,242,258 to Haselton and Alexander (Jun. 5,2001) entitled “Methods for the selective regulation of DNA and RNAtranscription and translation by photoactivation” and U.S. Pat. No.6,410,327 to Haselton, III, et al. entitled “Methods for the selectiveregulation of DNA and RNA transcription and translation byphotoactivation” describe DMNPE caging of DNA by postsyntheticreactions; Ando et al. (2001) “Photo-mediated gene activation usingcaged RNA/DNA in zebrafish embryos” Nature Genetics 28: 317-325describes Bhc caging of RNA and DNA by postsynthetic reactions; andChaulk and MacMillan (1998) “Caged RNA: Photo-control of a ribozymereaction” Nucl Acids Res. 26:3173-3178 describes 2-nitrobenzyl caging ofRNA by incorporation of a caged phosphoramidite during RNA synthesis. Acaged RNA or an RNA that is to be caged optionally includes one or moredeoxyribonucleotides and/or nonnatural or modified nucleotides, e.g.,that are less reactive than standard ribonucleotides, to facilitateattachment of the caging group(s), e.g., to a 5′ hydroxyl.

Caging groups can be attached at random and/or predetermined siteswithin a molecule. Useful site(s) of attachment of and/or conditions forattaching caging groups to a given molecule can be determined bytechniques known in the art. For example, a molecule with a knownactivity (e.g., an interfering RNA or a protein transduction domain) canbe reacted with a caging compound. The resulting caged molecule can thenbe tested to determine if its activity (e.g., ability to initiate RNAior to mediate introduction of an associated molecule into a cell) issufficiently abrogated. As another example, amino acid residues centralto the activity of a polypeptide (e.g., residues located at a bindinginterface of a protein transduction domain, or the like) can beidentified by routine techniques such as scanning mutagenesis, sequencecomparisons and site-directed mutagenesis, or the like. Such residuescan then be caged, and the activity of the caged polypeptide (e.g., itsability to mediate introduction of an associated molecule into a cell)can be assayed to determine the efficacy of caging. Similarly, an RNAcan be caged at positions and/or groups identified as being required foractivity (e.g., the 5′ phosphate or 5′ hydroxyl of the antisense strandof an siRNA can be caged).

An alternative method for caging a molecule (e.g., an siRNA) is toenclose the molecule in a photolabile vesicle (e.g., a photolabile lipidvesicle), optionally including a protein transduction domain or the like(FIG. 11). Similarly, the molecule can be loaded into the pores of aporous bead which is then encased in a photolabile gel.

Appropriate methods for uncaging caged molecules are also known in theart. For example, appropriate wavelengths of light for removing manyphotolabile groups have been described; e.g., 300-360 nm for2-nitrobenzyl, 350 nm for benzoin esters, and 740 nm for brominated7-hydroxycoumarin-4-ylmethyls (two-photon) (see, e.g., referencesherein). Conditions for uncaging any caged molecule (e.g., the optimalwavelength for removing a photolabile caging group) can be determinedaccording to methods well known in the art. Instrumentation and devicesfor delivering uncaging energy are likewise known (e.g., sonicators,heat sources, light sources, other sources of electromagnetic radiation,and the like). For example, well known and useful light sources includee.g., a lamp, a laser (e.g., a laser optically coupled to a fiber-opticdelivery system) or a light-emitting compound.

In vivo and in vitro Cellular Delivery

Molecules (e.g., double-stranded RNAs, including caged and/or labeledRNAs) can be introduced into cells by traditional methods such aslipofection, electroporation, microinjection, optofection, lasertransfection, calcium phosphate precipitation, and/or particlebombardment. Double-stranded RNA can also be introduced into cells bypinocytosis or by using streptolysin-O (SLO). See, e.g., WO 03/040375 byWolff entitled “Compositions and processes using siRNA, amphipathiccompounds and polycations.” Reagents for delivery of double-strandedRNAs are commercially available, e.g., TransIT-TKO™ (Mirus Corporation,www.genetransfer.com). If the molecule is caged, such delivery can beaccomplished without uncaging and thereby activating the molecule; forexample, a photoactivatable interfering RNA is not active during thedelivery process until exposed to light of appropriate wavelength.However, these methods require manipulation of the cells, e.g., addingand removing transfection materials, pre-treating cells, and specialapparatus and equipment, etc. In addition, some cells (particularlyprimary cells) are difficult to transfect by methods such aslipofection.

While the methods above are suitable for introducing molecules (e.g.,interfering RNAs and caged DNAs) into cells, this invention features asimpler and more effective method of introducing molecules into thecell. That is, the molecule is optionally associated (covalently ornon-covalently) with a cellular delivery module that can mediate itsintroduction into the cell. The cellular delivery module is typically,but need not be, a polypeptide, for example, a PEP-1 peptide, anamphipathic peptide, e.g., an MPG peptide (Simeoni et al. (2003)“Insight into the mechanism of the peptide-based gene delivery systemMPG: Implications for delivery of siRNA into mammalian cells” Nucl AcidsRes 31: 2717-2724), a cationic peptide (e.g., a homopolymer of lysine,histidine, or D-arginine), or a protein transduction domain (apolypeptide that can mediate introduction of a covalently associatedmolecule into a cell). See, e.g., Lane (2001) Bioconjugate Chem.,12:825-841; Bonetta (2002) The Scientist 16:38; and Curr Opin Mol Ther(2000) 2:162-7. For example, an interfering RNA (including a cagedand/or labeled interfering RNA) can be covalently associated with aprotein transduction domain (e.g., an HIV TAT sequence, which most cellsnaturally uptake, or a short D-arginine homopolymer, e.g., 8-D-Arg,eight contiguous D-arginine residues). The protein transductiondomain-coupled RNA can simply be, e.g., added to cell culture orinjected into an animal for delivery. (Note that TAT and D-argininehomopolymers, for example, can alternatively be noncovalently associatedwith the interfering RNA and still mediate its introduction into thecell.)

A number of polypeptides capable of mediating introduction of associatedmolecules into a cell are known in the art and can be adapted to thepresent invention; see, e.g., the references above and Langel (2002)Cell Penetrating Peptides CRC Press, Pharmacology & Toxicology Series.

As noted, an RNA, or a caged DNA, can also be introduced into cells bycovalently or noncovalently attached lipids, e.g., by a covalentlyattached myristoyl group. In any of the cellular delivery modulesherein, lipids used for lipofection are optionally excluded fromcellular delivery modules in some embodiments.

In summary, an RNA or a caged DNA can be introduced into a cell by anyof several methods, including without limitation, lipofection,electroporation, microinjection, and association with a cellulardelivery module (including covalent association with a proteintransduction domain). RNA and caged DNA can optionally be introducedinto specific tissues and/or cell types (e.g., explanted or in anorganism), for example, by laser transfection, gold particlebombardment, microinjection, coupling to viral proteins, or covalentassociation with a protein transduction domain, among other techniques.See, e.g., Robbins et al. (2002) “Peptide delivery to tissues viareversibly linked protein transduction sequences” Biotechniques33:190-192 and Rehman et al. (2003) “Protection of islets by in situpeptide-mediated transduction of the I-kappa B kinase inhibitorNemo-binding domain peptide” J Biol Chem 278:9862-9868.

The cell into which an RNA or a caged DNA of this invention isintroduced is typically a eukaryotic cell (e.g., a yeast, a vertebratecell, a mammalian cell, a rodent cell, a primate cell, a human cell, aplant cell, an insect cell, or essentially any other type of eukaryoticcell). The cell can be, e.g., in culture or in a tissue, fluid, etc.and/or from or in an organism.

The cellular delivery modules optionally can be caged. Covalentlyassociated cellular delivery modules (e.g., protein transductiondomains) can optionally be released from the associated molecule (e.g.,by placement of a photolabile linkage, a disulfide or ester linkage thatis reduced or cleaved in the cell, or the like, between the cellulardelivery module and the molecule). For example, 8-D-Arg can becovalently linked through a disulfide linker to an interfering RNA. The8-D-Arg module mediates entry of the RNA into a cell, where the linkeris reduced in the reducing environment of the cytoplasm, freeing theinterfering RNA from the 8-D-Arg module.

The amount of a nucleic acid delivered to a cell can optionally becontrolled by controlling the number of cellular delivery modulesassociated with the nucleic acid (covalently or noncovalently). Forexample, increasing the ratio of 8-D-Arg to interfering RNA can increasethe percentage of interfering RNA that enters the cell.

The RNAs and caged DNAs of this invention optionally also comprise asubcellular delivery module (e.g., a peptide, nucleic acid, and/orcarbohydrate tag) or other means of achieving a desired subcellularlocalization. For example, an interfering RNA is typically mosteffective at initiating RNAi when it is localized to the cytoplasm.Thus, if a method that results in localization of the interfering RNA tothe endosome is used to introduce the RNA into the cell (e.g.,lipofection, certain protein transduction domains, and the like),performance of the interfering RNA can be improved by including anendosomal release agent on the RNA (e.g., HA-2, PEI, or a dendrimer).See, e.g., Journal of Controlled Release (1999) 61:137-143; J Biol Chem277:27135-43; Proc Natl Acad Sci 89:7934-38; and Bioconjugate Chem(2002) 13:996-1001. Examples of subcellular delivery modules includenuclear localization signals, chloroplast stromal targeting sequences,and many others (see, e.g., Molecular Biology of the Cell (3rd ed.)Alberts et al., Garland Publishing, 1994; and Molecular Cell Biology(4th ed.) Lodish et al., W H Freeman & Co, 1999). Similarly,localization can be to a target protein; that is, the subcellulardelivery module can comprise a binding domain that binds the targetprotein.

Labels

The compositions of this invention optionally include one or morelabels; e.g., optically detectable labels, such as fluorescent orluminescent labels, and/or non-optically detectable labels, such asmagnetic labels. A number of fluorescent labels are well known in theart, including but not limited to, quantum dots, hydrophobicfluorophores (e.g., coumarin, rhodamine and fluorescein), and greenfluorescent protein (GFP) and variants thereof (e.g., cyan fluorescentprotein and yellow fluorescent protein). See e.g., Haughland (2002)Handbook of Fluorescent Probes and Research Products, Ninth Edition orthe current Web Edition, both available from Molecular Probes, Inc.Likewise, a variety of donor/acceptor and fluorophore/quenchercombinations, using e.g., fluorescence resonance energy transfer(FRET)-based quenching, non-FRET based quenching, or wavelength-shiftingharvester molecules, are known. Example combinations include cyanfluorescent protein and yellow fluorescent protein, terbium chelate andTRITC (tetrarhodamine isothiocyanate), lanthanide (e.g., europium orterbium) chelates and allophycocyanin (APC) or Cy5, europium cryptateand Allophycocyanin, fluorescein and tetramethylrhodamine, IAEDANS andfluorescein, EDANS and DABCYL, fluorescein and DABCYL, fluorescein andfluorescein, BODIPY FL and BODIPY FL, and fluorescein and QSY 7 dye.Nonfluorescent acceptors such as DABCYL and QSY 7 and QSY 33 dyes havethe particular advantage of eliminating background fluorescenceresulting from direct (i.e., nonsensitized) acceptor excitation. See,e.g., U.S. Pat. Nos. 5,668,648, 5,707,804, 5,728,528, 5,853,992, and5,869,255 to Mathies et al. for a description of FRET dyes.

For use of quantum dots as labels for biomolecules, see, e.g., Dubertretet al. (2002) Science 298:1759; Nature Biotechnology (2003) 21:41-46;and Nature Biotechnology (2003) 21:47-51. In the context of the presentinvention, such quantum dots can be used to label any nucleic acid ofinterest, e.g., an interfering RNA, e.g., a caged interfering RNA.

Other optically detectable labels can also be used in the invention. Forexample, gold beads can be used as labels and can be detected using awhite light source via resonance light scattering. See, e.g.,http://www.geniconsciences.com. Suitable non-optically detectable labelsare also known in the art. For example, magnetic labels can be used inthe invention (e.g., 3 nm superparamagnetic colloidal iron oxide as alabel and NMR detection; see e.g., Nature Biotechnology (2002)20:816-820).

Labels can be introduced to nucleic acids during synthesis or bypostsynthetic reactions by techniques established in the art. Forexample, a fluorescently labeled nucleotide can be incorporated into anRNA or DNA during enzymatic or chemical synthesis of the nucleic acid,e.g., at a preselected or random nucleotide position. Alternatively,fluorescent labels can be added to RNAs or DNAs by postsyntheticreactions, at either random or preselected positions (e.g., anoligonucleotide can be chemically synthesized with a terminal amine orfree thiol at a preselected position, and a fluorophore can be coupledto the oligonucleotide via reaction with the amine or thiol). Reagentsfor fluorescent labeling of nucleic acids are commercially available;for example, a variety of kits for fluorescently labeling nucleic acidsare available from Molecular Probes, Inc. (www.probes.com), and a kitfor randomly labeling double-stranded RNA is available from Ambion, Inc.(www.ambion.com, the Silencer™ siRNA labeling kit). Quenchers can beintroduced by analogous techniques.

Attachment of labels to oligos during automated synthesis and bypost-synthetic reactions has been described. See, e.g., Tyagi and Kramer(1996) “Molecular beacons: probes that fluoresce upon hybridization”Nature Biotechnology 14:303-308; U.S. Pat. No. 6,037,130 to Tyagi et al.(Mar. 14, 2000), entitled “Wavelength-shifting probes and primers andtheir use in assays and kits”; and U.S. Pat. No. 5,925,517 (Jul. 20,1999) to Tyagi et al. entitled “Detectably labeled dual conformationoligonucleotide probes, assays and kits.” Additional details onsynthesis of functionalized oligos can be found in Nelson, et al. (1989)“Bifunctional Oligonucleotide Probes Synthesized Using A Novel CPGSupport Are Able To Detect Single Base Pair Mutations” Nucleic AcidsResearch 17:7187-7194.

Labels and/or quenchers can be introduced to the oligonucleotides, forexample, by using a controlled-pore glass column to introduce, e.g., thequencher (e.g., a 4-dimethylaminoazobenzene-4′-sulfonyl moiety (DABSYL).For example, the quencher can be added at the 3′ end of oligonucleotidesduring automated synthesis; a succinimidyl ester of4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL) can be used when thesite of attachment is a primary amino group; and4-dimethylaminophenylazophenyl-4′-maleimide (DABMI) can be used when thesite of attachment is a sulfhydryl group. Similarly, fluorescein can beintroduced into oligos, either using a fluorescein phosphoramidite thatreplaces a nucleoside with fluorescein, or by using a fluorescein dTphosphoramidite that introduces a fluorescein moiety at a thymidine ringvia a spacer. To link a fluorescein moiety to a terminal location,iodoacetoamidofluorescein can be coupled to a sulfhydryl group.Tetrachlorofluorescein (TET) can be introduced during automatedsynthesis using a 5′-tetrachloro-fluorescein phosphoramidite. Otherreactive fluorophore derivatives and their respective sites ofattachment include the succinimidyl ester of 5-carboxyrhodamine-6G (RHD)coupled to an amino group; an iodoacetamide of tetramethylrhodaminecoupled to a sulfhydryl group; an isothiocyanate of tetramethylrhodaminecoupled to an amino group; or a sulfonylchloride of Texas red coupled toa sulfhydryl group. Labeled oligonucleotides can be purified, ifdesired, e.g., by high pressure liquid chromatography or other methods.

Similarly, signals from the labels (e.g., absorption by and/orfluorescent emission from a fluorescent label) can be detected byessentially any method known in the art. For example, multicolordetection, detection of FRET (including, e.g., time-resolved or TR-FRET,e.g., between lanthanide chelate donors and fluorescent dye acceptors;see, e.g., Journal of Biomolecular Screening (2002) 7:3-10), and thelike, are well known in the art. In brief, FRET (Fluorescence ResonanceEnergy Transfer) is a non-radiative energy transfer phenomenon in whichtwo fluorophores with overlapping emission and excitation spectra, whenin sufficiently close proximity, experience energy transfer by aresonance dipole induced dipole interaction. The phenomenon is commonlyused to study the binding of analytes such as nucleic acids, proteinsand the like. FRET is a distance dependent excited state interaction inwhich emission of one fluorophore is coupled to the excitation ofanother which is in proximity (close enough for an observable change inemissions to occur). Some excited fluorophores interact to formexcimers, which are excited state dimers that exhibit altered emissionspectra (e.g., phospholipid analogs with pyrene sn-2 acyl chains); see,e.g., Haughland (2003) Handbook of Fluorescent Probes and ResearchProducts Ninth Edition, available from Molecular Probes. Astraightforward discussion of FRET can be found in the Handbook and thereferences cited therein.

As another example, fluorescence polarization can be used. Briefly, inthe performance of such fluorescent binding assays, a typically small,fluorescently labeled molecule, e.g., a ligand, antigen, etc., having arelatively fast rotational correlation time, is used to bind to a muchlarger molecule, e.g., a receptor protein, antibody etc., which has amuch slower rotational correlation time. The binding of the smalllabeled molecule to the larger molecule significantly increases therotational correlation time (decreases the amount of rotation) of thelabeled species, namely the labeled complex over that of the freeunbound labeled molecule. This has a corresponding effect on the levelof polarization that is detectable. Specifically, the labeled complexpresents much higher fluorescence polarization than the unbound, labeledmolecule.

Generally, fluorescence polarization level is calculated using thefollowing formula:P=[I ₁ −I ₂ ]/[I ₁ +I ₂]where I₁ is the fluorescence detected in the plane parallel to theexcitation light, and I₂ is the fluorescence detected in the planeperpendicular to the excitation light. References which discussfluorescence polarization and/or its use in molecular biology includePerrin (1926) “Polarization de la lumiere de fluorescence. Vie moyennede molecules dans l'etat excite” J Phys Radium 7:390; Weber (1953)“Rotational Brownian motion and polarization of the fluorescence ofsolutions” Adv Protein Chem 8:415; Weber (1956) J Opt Soc Am 46:962;Dandliker and Feigen (1961) “Quantification of the antigen-antibodyreaction by the polarization of fluorescence” Biochem Biophys Res Commun5:299; Dandliker and de Saussure (1970) “Fluorescence polarization inimmunochemistry” Immunochemistry 7:799; Dandliker et al. (1973)“Fluorescence polarization immunoassay. Theory and experimental method”Immunochemistry 10:219; Levison et al. (1976) “Fluorescence polarizationmeasurement of the hormone-binding site interaction” Endocrinology99:1129; Jiskoot et al. (1991) “Preparation and application of afluorescein-labeled peptide for determining the affinity constant of amonoclonal antibody-hapten complex by fluorescence polarization” AnalBiochem 196:421; Wei and Herron (1993) “Use of synthetic peptides astracer antigens in fluorescence polarization immunoassays of highmolecular weight analytes” Anal Chem 65:3372; Devlin et al. (1993)“Homogeneous detection of nucleic acids by transient-state polarizedfluorescence” Clin Chem 39:1939; Murakami et al. (1991)Fluorescent-labeled oligonucleotide probes detection of hybrid formationin solution by fluorescence polarization spectroscopy” Nuc. Acids Res19:4097; Checovich et al. (1995) “Fluorescence polarization-a new toolfor cell and molecular biology” Nature 375:354-256; Kumke et al. (1995)“Hybridization of fluorescein-labeled DNA oligomers detected byfluorescence anisotropy with protein binding enhancement” Anal Chem67:21, 3945-3951; and Walker et al. (1996) “Strand displacementamplification (SDA) and transient-state fluorescence polarizationdetection of mycobacterium tuberculosis DNA” Clinical Chemistry 42:1,9-13.Arrays

In certain embodiments, the RNA is arranged in an array. In an array ona matrix (e.g., a surface), each nucleic acid is bound (e.g.,electrostatically or covalently bound, directly or via a linker) to thematrix at a unique location. Methods of making, using, and analyzingsuch arrays (e.g., microarrays) are well known in the art, includingmethods of using arrays by overlaying the arrays with cells into whichthe components of the array can be introduced. See e.g., U.S. Pat. No.6,197,599; Ziauddin and Sabatini “Microarrays of cells expressingdefined cDNAs” Nature May 3, 2001;411(6833):107-10; and Falsey et al.Bioconjug. Chem. (2001) 12:346-53.

Molecular Biological Techniques

In practicing the present invention, many conventional techniques inmolecular biology, microbiology, and recombinant DNA technology areoptionally used (e.g., for making and/or manipulating nucleic acids,polypeptides, and/or cells of the invention). These techniques are wellknown, and detailed protocols for numerous such procedures (including,e.g., in vitro amplification of nucleic acids, cloning, mutagenesis,transformation, cellular transduction with nucleic acids, proteinexpression, and/or the like) are described in, for example, Berger andKimmel, Guide to Molecular Cloning Techniques, Methods in Enzymologyvolume 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook etal., Molecular Cloning—A Laboratory Manual (3rd Ed.), Vol. 1-3, ColdSpring Harbor Laboratory, Cold Spring Harbor, New York, 2002(“Sambrook”) and Current Protocols in Molecular Biology, F. M. Ausubelet al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (supplementedthrough 2004) (“Ausubel”)). Other useful references, e.g. for cellisolation and culture (e.g., for subsequent nucleic acid or proteinisolation) include Freshney (1994) Culture of Animal Cells, a Manual ofBasic Technique, third edition, Wiley-Liss, New York and the referencescited therein; Payne et al (1992) Plant Cell and Tissue Culture inLiquid Systems John Wiley & Sons, Inc. New York, N.Y.; Gamborg andPhillips (Eds.) (1995) Plant Cell, Tissue and Organ Culture; FundamentalMethods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg NewYork) and Atlas and Parks (Eds.) The Handbook of Microbiological Media(1993) CRC Press, Boca Raton, Fla.

Oligonucleotide Synthesis

In general, synthetic methods for making oligonucleotides and PNAs(including labeled oligos and PNAs) are well known. For example,oligonucleotides can be synthesized chemically according to the solidphase phosphoramidite triester method described by Beaucage andCaruthers (1981), Tetrahedron Letts., 22(20):1859-1862, e.g., using acommercially available automated synthesizer, e.g., as described inNeedham-VanDevanter et al. (1984) Nucleic Acids Res., 12:6159-6168.Synthesis of PNAs and modified oligonucleotides (e.g., oligonucleotidescomprising 2′-O-methyl nucleotides and/or phosphorothioate,methylphosphonate, or boranophosphate linkages) are described in e.g.,Oligonucleotides and Analogs (1991), IRL Press, New York; Shaw et al.(1993), Methods Mol. Biol. 20:225-243; Nielsen et al. (1991), Science254:1497-1500; and Shaw et al. (2000) Methods Enzymol. 313:226-257.

Oligonucleotides, including modified oligonucleotides (e.g.,oligonucleotides comprising fluorophores and quenchers, unnaturalnucleotides, 2′-O-methyl nucleotides, and/or phosphorothioate,methylphosphonate, or boranophosphate linkages) can also be ordered froma variety of commercial sources known to persons of skill. There aremany commercial providers of oligo synthesis services, and thus, this isa broadly accessible technology. Any nucleic acid can be custom orderedfrom any of a variety of commercial sources, such as The MidlandCertified Reagent Company (www.mcrc.com), The Great American GeneCompany (www.genco.com), ExpressGen Inc. (www.expressgen.com), QIAGEN(http://oligos.qiagen.com), Dharmacon (www.dharmacon.com), and manyothers.

A variety of nuclease-resistant nucleic acids can optionally be created,e.g., comprising modified nucleotides and/or modified internucleotidelinkages such as those currently used in the synthesis of antisenseoligonucleotides. For example, a nuclease resistant oligonucleotide cancomprise one or more 2′-O-methyl nucleotides. For example, anoligonucleotide comprising standard deoxyribonucleotides can alsocomprise one or more 2′-O-methyl nucleotides (e.g., at its 5′ end), oran oligonucleotide can consist entirely of 2′-O-methyl nucleotides. Asanother example, a nuclease resistant oligonucleotide can comprise oneor more phosphorothioate linkages (oligonucleotides comprising suchlinkages are sometimes called “S-oligos”). An oligonucleotide cancomprise, e.g., only phosphorothioate linkages or a mixture ofphosphodiester and phosphorothioate linkages. In other embodiments, theoligonucleotide comprises one or more methylphosphonate linkages, one ormore boranophosphate linkages, or the like. Combinations of typicalnuclease resistance modification strategies can also be employed; forexample, a nuclease resistant oligonucleotide can comprise both2′-O-methyl nucleotides and phosphorothioate linkages.

As noted, a nucleic acid can be produced by chemical synthesis or can becustom ordered. In addition, nucleic acids can be produced by enzymaticsynthesis (in vitro or in vivo). For example, interfering RNAs can beproduced by in vitro transcription using techniques well known in theart. Kits for in vitro transcription are commercially available; forexample, the Silencer™ siRNA construction kit from Ambion, Inc.(www.ambion.com).

Polypeptide Production

Polypeptides (e.g., polypeptide cellular delivery modules, e.g., proteintransduction domains) can optionally be produced by expression in a hostcell transformed with a vector comprising a nucleic acid encoding thedesired polypeptide(s). Expressed polypeptides can be recovered andpurified from recombinant cell cultures by any of a number of methodswell known in the art, including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography (e.g., using any of the tagging systems notedherein), hydroxylapatite chromatography, and lectin chromatography, forexample. Protein refolding steps can be used, as desired, in completingconfiguration of the mature protein. Finally, high performance liquidchromatography (HPLC) can be employed in the final purification steps.See, e.g., the references noted above and Deutscher, Methods inEnzymology Vol. 182: Guide to Protein Purification, Academic Press, Inc.N.Y. (1990); Sandana (1997) Bioseparation of Proteins, Academic Press,Inc.; Bollag et al. (1996) Protein Methods, 2^(nd) Edition Wiley-Liss,NY; Walker (1996) The Protein Protocols Handbook Humana Press, NJ;Harris and Angal (1990) Protein Purification Applications: A PracticalApproach IRL Press at Oxford, Oxford, U.K.; Scopes (1993) ProteinPurification: Principles and Practice 3^(rd) Edition Springer Verlag,NY; Janson and Ryden (1998) Protein Purification: Principles, HighResolution Methods and Applications, Second Edition Wiley-VCH, NY; andWalker (1998) Protein Protocols on CD-ROM Humana Press, NJ.

Alternatively, cell-free transcription/translation systems can beemployed to produce polypeptides encoded by nucleic acids. A number ofsuitable in vitro transcription and translation systems are commerciallyavailable. A general guide to in vitro transcription and translationprotocols is found in Tymms (1995) In vitro Transcription andTranslation Protocols: Methods in Molecular Biology Volume 37, GarlandPublishing, NY.

In addition, polypeptides (including, e.g., polypeptides comprisingfluorophores and quenchers and/or unnatural amino acids) can be producedmanually or by using an automated system, by direct peptide synthesisusing solid-phase techniques (see, e.g., Stewart et al. (1969)Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco; MerrifieldJ (1963) J. Am. Chem. Soc. 85:2149-2154). Exemplary automated systemsinclude the Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer,Foster City, Calif.). In addition, there are many commercial providersof peptide synthesis services. If desired, subsequences can bechemically synthesized separately, and combined using chemical methodsto provide full-length polypeptides.

EXAMPLES

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. Accordingly, the following examples areoffered to illustrate, but not to limit, the claimed invention.

PA Sensors: Constructs and Methods for Measuring RNA Transcripts inLiving Cells

In one aspect, the present invention provides sensors for detecting andmeasuring mRNA in living cells (also known as PAC probes for mRNA) andmethods of controlling activation of such mRNA sensors in living cells.In one class of embodiments, the sensor is attached to one or morephoto-labile groups that protect the sensor from extra-cellular andintra-cellular degradation and, at the same time, inactivate the sensor.Upon exposure to light of a specific wavelength, the photolabile groupsdetach from the sensor and the sensor becomes active. The mRNA sensorsinclude one or more labels (e.g., a combination of acceptor and donorfluorophores that interact via FRET or ET or a fluorophore/quenchercombination) on RNAs that can initiate RNAi (e.g., siRNA, shRNA; FIGS.12-14, in which A and B represent either a fluorescent label and aquencher (or vice versa) or a donor and acceptor (or vice versa)). Thesignal from the sensor is used to detect and measure mRNA in livingcells. Splice variants of mRNAs, for example, can also be analyzed usinginterfering RNA approaches.

Traditional or novel delivery methods can be used to introduce asufficient quantity of mRNA sensors into cells. A high throughputuncaging device, such as those described in U.S. Ser. No. 60/427,664filed Nov. 18, 2002, 60/436,855 filed Dec. 26, 2002, 60/439,917 filedJan. 13, 2003, 60/451,177 filed February 27, and 60/456,870 filed Mar.21, 2003, can be used to activate photoactivatable sensors, e.g., incells grown in a microtiter plate. This invention also features methodsof detecting and measuring mRNA with such sensors in living cells.

The ability to monitor immediate changes in mRNA levels in living cellsfacilitates the development of a broad range of cell-based assays forbasic research, pharmaceutical industries, clinical and agriculturaldiagnostics. For example, a specific GPCR or kinase cell-based assay canbe developed for screening lead compounds using one or more PAC probesfor monitoring mRNAs downstream of the GPCR or kinase. Actual transcriptor surrogate transcript (marker, mRNA of a gene further downstream in apathway) response to modulation of specific pathways by the compoundscan be monitored in living cells.

An example PAC probe for an mRNA comprises a labeled interfering RNA(e.g., an siRNA or a shRNA; see, e.g., Watanabe (Jan. 13, 2003)Scientist 17(1):36; D Engelke (2002) Nature Biotech 29: 505; Trends inBiotech 20:49 (2002)); one or more caging groups, e.g., photolabilecaging groups (see, e.g., F R Haselton JBC 274:20895 and H Okamoto(2001) Nature Genetics 28:317); and optionally a cellular and/orsubcellular delivery module, e.g., a peptide delivery module such as TATor Antp (see, e.g., Lane (2001) Bioconjugate Chemistry 12:825).

Modified nucleotides can optionally be incorporated into interferingRNAs to reduce degradation in cells. For example, a phosphate backboneanalog (e.g., phosphorothioate and/or a modified nucleotide (forexample, a 2′-O-methyl nucleotide, e.g., 2′-O-methylinosine) can be usedto protect the RNA from nuclease digestion. Caging groups can alsoprotect against nuclease digestion.

FIG. 12 describes a small interfering RNA (siRNA, also known as shortinterfering RNA) structure used for detecting mRNA in living cells. ThesiRNA can be, for example, a 21-25 mer double-stranded RNA; otherlengths and/or optional overhangs (e.g., two nucleotide 3′ overhangs)can also be used. A reporter combination (e.g., a fluorophore/quencherpair or acceptor/donor FRET pair) is linked at the 5′ and 3′ ends of onestrand or at the ends of opposite strands. The reporter molecules canalso be within the siRNA, either on the same strand or on oppositestrands of the double stranded siRNA. The reporters can be, e.g., acombination of FRET dyes such as coumarin and FITC or a combination suchas europium and APC that permits application of time-resolvedfluorescence (TRF) techniques.

An interfering RNA can be caged, e.g., with photo-labile groups, at thephosphates, riboses and/or bases to protect it and to inactivate itsfunction. It can optionally be linked to a delivery module, e.g., apeptide delivery module (for example, 8-D-Arg, Antp, Pep-1, or thelike), e.g., with a disulfide linker as illustrated in FIG. 14. Otherestablished delivery approaches can also be used, e.g., lipofection.

FIG. 13 shows another type of interfering RNA for measuring mRNA, e.g.,a short hairpin RNA (shRNA, also called small hairpin RNA; e.g., NatureGenetics 33:396). For example, a shRNA can have about 60-70 nt that forma hairpin, e.g., with a 25-30 mer double-stranded region and an 8 mersingle-stranded loop. A reporter combination (e.g., a donor and anacceptor fluorophore that interact via FRET, or a fluorophore/darkquencher) can be attached for signaling the presence of a specific RNAtranscript. As in the previous example, the shRNA can be caged, e.g.,with photolabile caging groups.

FIG. 15 shows the detection of mRNA using an interfering RNA PAC probe.The siRNA is incorporated into the RISC complex, and the antisensestrand guides cleavage of the target mRNA (promoting its degradation).Strand separation of the interfering RNA probe leads to the separationof the reporter molecules on the RNA, resulting in a detectable signalor change in signal (as indicated by the starburst symbol). MultiplemRNA transcripts can be analyzed using interfering RNAs with differentreporter molecules (e.g., fluorophores that emit at differentwavelengths).

FIG. 16 shows the detection of a single target using multiple (e.g., twoor more) interfering RNA sensors. The different interfering RNAstypically emit distinguishable signals before and/or after initiation ofRNAi. Detection specificity is improved using this design, because anactual signal or signal change (indicating degradation of the specifictarget mRNA) is recorded only when signals from both interfering RNAsare observed at about the same time.

Applications

FIG. 17 shows an example workflow for mRNA measurement using the sensorsof this invention, where the effect of a compound (drug, agonist,antagonist, etc. affecting or potentially affecting an upstreamsignaling molecule) on mRNA level is monitored. There are minimalfluidic handling steps and reagents required. A photolabile PAC probecan be uncaged by exposing to a light source (e.g., in an uncagingdevice such as those described in U.S. Ser. No. 60/427,664 filed Nov.18, 2002, 60/436,855 filed Dec. 26, 2002, 60/439,917 filed Jan. 13,2003, 60/451,177 filed February 27, and 60/456,870 filed Mar. 21, 2003).

A PAC probe for mRNA can be used to measure amount of mRNA transcriptand location of mRNA processing in living cells. When performingquantitative analysis, an interfering RNA sensor for a house-keeping RNAcan optionally be used to normalize for variable target(s). Deviationbetween different cells can be corrected if one or more dual-labeledFRET interfering RNAs, for example, are used instead of a darkquencher/fluorophore probe format. With a dual-labeled FRET probe (i.e.,a probe with a donor fluorophore and an acceptor fluorophore, where thedonor and acceptor are capable of exhibiting FRET), at least twodifferent signals can be obtained, i.e., the FRET signal (emission bythe acceptor following stimulation of the donor) and the acceptor signal(emission by the acceptor following stimulation of the acceptor) usingdifferent excitation wavelengths, e.g., produced by different lasers, tostimulate the donor and acceptor. The ratio of these two signals can betaken, e.g., to normalize for transfection efficiency of the probe.

As noted, interfering RNA PAC probes can be used to analyze splicevariants (including, e.g., in living cells). Examples of genes with avariety of splice variants are beta-actin and cyclic nucleotidephosphodiesterases (Current Opinion in Cell Biology (2000) 12:174-179),among many others. To analyze alternatively spliced mRNAs, for example,a siRNA probe can be designed to recognize the splice junction. One ormore such siRNAs can be used to detect various isoforms. For example,FIG. 18 illustrates how multiple siRNAs can be used to determine splicevariants. A nuclear RNA containing three exons and two introns istranscribed from chromosomal DNA. The nuclear RNA is spliced to form themRNA, which in this example includes all three exons and no introns. AsiRNA is designed to be at the splice junction. The isoform with thecorrect splice variant is digested. Similarly, siRNA can be made to bindto the exon regions and not between the splice junctions, or a siRNA canbe designed against an intron. Splice variants containing the intron aredigested and result in a signal from the siRNA probe.

Cell Based Assay using Labeled Interfering RNA as in vivo mRNA Sensor

The following sets forth a series of experiments that demonstrate designand use of interfering RNA sensors to detect GAPDH mRNA. GAPDH isconstitutively expressed.

Three different interfering RNAs were designed against GAPDH (FIG. 19;SEQ ID NO:1): RNAi 1, corresponding to nt 690-708 (each strand has 19GAPDH bases plus a TT 3′ overhang), RNAi 2, corresponding to nt 915-936(each strand has 21 GAPDH bases, forming a 19 bp double-stranded regionand two nucleotide overhangs), and RNAi 3, corresponding to nt 601-621(each strand has 21 GAPDH bases, forming a 19 bp double-stranded regionand two nucleotide overhangs). Each RNA was labeled with 6-FAM on the 3′end of the antisense strand, and a Dabcyl quencher was attached to the3′ end of the sense strand. The FAM label and Dabcyl quencher wereincorporated during oligonucleotide synthesis. FIG. 20 illustrates oneof the three GAPDH RNAi sensors. When the sense and antisense strandsare annealed, the FAM label is quenched (Panel A); when the strands aredenatured, the label is not quenched and fluoresces (Panel B). Panel Cshows fluorescent emission spectra for the antisense strand (curve 1),the sense strand (curve 2), and the annealed strands (curve 3),illustrating that the FAM label is quenched in the annealed sensor.

To verify that the labeled RNAs were able to attenuate expression ofGAPDH, labeled RNAi 1-3 were lipofected into HeLa cells (1000 cells) ata concentration of 0.5 μg/μl for 4 hours. Cells were maintained at twotemperatures (37° C. and 45° C.) and lysed at different time pointsafter lipofection (4 h, 10 h, 20 h, 34 h, and 44 h). GAPDH mRNA wasmeasured using a branched DNA (bDNA) assay (see, e.g., Journal ofClinical Virology (2002) 25:205-216; QuantiGene bDNA assay kits arecommercially available from Genospectra, Inc., www.genospectra.com).

FIG. 21 shows the GAPDH mRNA level as measured by the bDNA assay at theindicated time points after lipofection of labeled RNAi 1 (Panel A), ascompared to a negative control (Panel B, no lipofection reagent). FIG.22 compares the percentage knockout of GAPDH expression, as measured bythe bDNA assay, for labeled RNAi 1-3. RNAi 1 was the most potentsilencer of the three interfering RNAs tested, knocking out GAPDHexpression in HeLa cells by as much as 90%.

To test the labeled RNAi's as in vivo mRNA sensors, the three GAPDHRNAis (labeled with 6-FAM and Dabcyl) were lipofected into HeLa cells(1000 cells) at a concentration of 0.5 μg/μl for 4 h at 37° C. The cellswere incubated with fresh medium at 37° C. Cells were fixed 4 h and 20 hafter lipofection and scanned on a Packard scanner for FITC signal, andbDNA assays were performed at the same time points. FIG. 23 shows theresults of the bDNA assays (RLU, luminescence) compared to the FITCsignals (FLU) for cells lipofected with the RNAi 1 (Panel A), RNAi 2(Panel B), and RNAi 3 (Panel C) sensors. We observed opposing trendsover time between the signals for the labeled RNAi sensors (increasedFITC signal, reflecting degradation of GAPDH mRNA) and the bDNA data(reduced GAPDH mRNA level in the presence of interfering RNA).

FIG. 24 shows the ratio of the bDNA assay measurement of GAPDH mRNAlevels at 20 h/4 h and the ratio of the FITC signal from labeled RNAi's1-3 at 20 h/4 h, and demonstrates that RNAi 1 is the most effective insilencing GAPDH and the most prominent in generating FRET signal.

Labeled RNAi 1 was further tested as an in vivo mRNA sensor. 2000 HeLacells were plated in each well in eight well chambers with complete DMEMmedium overnight at 37° C. Medium was changed to OptiMEM, and the cellswere lipofected with GAPDH RNAi 1 (2 μg, 4 μg) for 4 h in reduced serummedium at 37° C. At three different time points (0 h, 4 h, and 10 hafter lipofection), duplicate slides were plated. One slide was used fora bDNA assay, the other for scanning the FAM signal. For the bDNA assay,cells were lysed with bDNA lysis buffer at 0 h, 4 h, and 10 h timepoints, and lysate from approximately 300 cells was assayed for GAPDHmRNA using the bDNA assay. (Note that “0 h” is the time point after thelipofection process, which takes about 4 hours.) At each time point, theduplicate slide was fixed and scanned on a Packard scanner in the FAMchannel at 90% power and 70% PMT gain.

Fluorescent signal from the RNAi 1 sensor increased over time (from 0 to4 h and from 4 to 10 h) at both amounts of sensor tested (data notshown). Fluorescent signal from the sensor also increased withincreasing amount of sensor; 4 μg of RNAi 1 produced a more intensesignal than 2 μg at each time point. At both amounts of RNAi 1 tested (2μg and 4 μg), the level of GAPDH mRNA as measured by the bDNA assaytypically decreased over time.

An additional test of labeled RNAi 1 as an in vivo mRNA sensor wasperformed. 2000 HeLa cells were plated in each well in eight well glassslides with complete DMEM medium overnight at 37° C. Medium was changedto OptiMEM, and the cells were lipofected with 4 μg of GAPDH RNAi 1 for4 h in reduced serum medium at 37° C. At three different time points (0h, 4 h, and 10 h after lipofection), duplicate slides were plated. Oneslide was used for a bDNA assay, the other for scanning the FAM signal.For the bDNA assay, cells were lysed with bDNA lysis buffer at 0 h, 4 h,and 10 h time points, and lysate from approximately 350 cells wasassayed for GAPDH mRNA using the bDNA assay. (Note that “0 h” is thetime point after the lipofection process, which takes about 4 hours.) Ateach time point, the duplicate slide was fixed and scanned on a Packardmicroarray scanner in the FAM channel at 90% power and 60% PMT gain; foreach well, fluorescent signal from the entire well was analyzed. Theexperiment was performed in duplicate on three independent days, and theresulting data were averaged to obtain the results plotted in FIG. 25.

FIG. 25 Panel A shows the results of the bDNA assay (diamonds, RLU,representing the GAPDH mRNA level in the cells at the indicated timepoints) and the fluorescent signal for the labeled RNAi 1 mRNA PAC probe(circles, RFU), at each time point. FIG. 25 Panel B plots thefluorescent signals from the labeled RNAi 1 sensor against the resultsof the bDNA assay. We note an inverse linear relationship between thesignal from the interfering RNA sensor and the amount of GAPDH mRNAremaining in the cells.

It is worth noting that in the examples above, the level of fluorescentsignal from the siRNA sensor is correlated to the cumulative destructionof GAPDH mRNA in the cells. As more GAPDH mRNA gets degraded, the signalfrom the sensor increases. (Clearly, in these examples, the increase insensor signal level from 0 h to 4 h to 10 h (e.g., for the RNAi 1 sensorin FIG. 25) does not mean that the GAPDH mRNA level is increasing.)Therefore, for transcripts already abundant in cells (e.g.,constitutively expressed genes, such as GAPDH), an siRNA sensor canprovide an indication of the knock-down efficiency of the siRNA. Themethods can similarly be applied to determine the knock-down efficiencyof an siRNA against an inducible target mRNA.

In summary, we conclude from the above experiments that the magnitude ofthe FRET signal for the FAM label on the RNA sensor correlates to thelevel of GAPDH expression knockdown as measured by the bDNA assay andinversely correlates with the level of GAPDH remaining in the cell,confirming that labeled GAPDH interfering RNA functions as an inhibitorsensor.

Discussion

It will be evident to one of skill that the methods of detecting targetmRNA in a cell using a labeled interfering RNA sensor described hereinhave a number of applications, and that the signal output detected fromthe sensor can provide different types of information under differentcircumstances. The signal output is typically proportional to the amountof target mRNA degraded; depending on the circumstances, the signaloutput can be, e.g., proportional to the amount of target mRNA initiallypresent or induced in the cell and/or inversely proportional to theamount of mRNA remaining in the cell, as illustrated in the followingexamples.

For example, the methods can be used to determine how effective anygiven siRNA is at knocking down (or knocking out) expression of itstarget mRNA, e.g., in real time in living cells. The siRNA can belabeled to produce an siRNA sensor, which can be used in the methodsdescribed herein. For example, in the experiments described above, thesignal output from RNAi 1 is stronger than that from RNAi 2, indicatingthat RNAi 1 leads to the degradation of more GAPDH mRNA then RNAi 2, andthus indicating that RNAi 1 is better at knocking down GAPDH expressionthen is RNAi 2 (see, e.g., FIGS. 22-24).

As another example, the methods can be used for real-time, dynamicmonitoring of target mRNA levels. The experiment summarized in FIG. 25,for example, illustrates an inverse linear relationship between targetmRNA levels and signal output from the sensor. In this example, astronger fluorescent signal from the sensor indicates more of theconstitutively expressed GAPDH transcript has been degraded and thusthat less of the transcript is currently present in the cell.

The methods can be used to monitor both constitutively expressed and/orinducible target mRNA levels. Thus, in yet another example, the methodscan be used to detect expression of an inducible gene, e.g., in realtime in living cells. For example, an siRNA sensor for an inducibletarget gene (e.g., IL-8) can be introduced into cells, expression of thetarget gene can be induced, and the level of signal from the siRNAsensor (e.g., the slope, intercept(s), and/or maximum value(s) from aplot of signal strength versus time) can be used as an indication of theonset of target gene expression and/or the degree of induction of thetarget gene. In this example, the level of fluorescent signal from thesiRNA sensor is correlated to the degree of induction. As more targetgene transcript becomes available for RNA interference, the sensorsignal increases. Again, the level of sensor signal reflects the amountof transcript being destroyed and not the final level of the inducibletranscript. Since the amount of transcript destroyed is proportional tothe degree of induction of the inducible gene, the level of sensorsignal is proportional to the degree of induction: a stronger signalindicates stronger induction (more transcripts destroyed).

As yet another example, a caged siRNA sensor can be used in the methodsto detect the mRNA level of a target gene, e.g., in real time in livingcells. The caged siRNA sensor is put into the cells and is then uncaged(e.g., at a preselected time). The level of signal from the sensor canbe used as a measurement of the transcript level immediately prior touncaging. Preferably, in these example embodiments, the concentration ofthe caged siRNA sensor is higher than the concentration of the targetmRNA. The slope, intercept(s), and/or maximum value(s) from a plot ofsignal strength versus time after uncaging, for example, can be used toreflect the target mRNA level at the time of uncaging. Again in thisexample, a stronger signal from the sensor indicates degradation of moretarget mRNA and thus a higher concentration of the target mRNA in thecell at the time of uncaging.

In vivo Photoactivation of Photolabile Caged siRNA

The following sets forth a series of experiments that demonstrate use ofa photolabile caged siRNA to control initiation of RNAi of the GAPDHmRNA.

In vivo Photoactivation

The 5′ phosphate of the antisense strand of RNAi 1 was caged (FIG. 28).The caged antisense oligo (5′ PhotoCageAGUAGAGGCAGGGAUGAUGdTdT 3′, SEQID NO:2) was synthesized by Trilink Biotechnologies, Inc.(www.trilinkbiotech.com), as follows. The commercially available cagedphosphoramidite[1-N-(4,4′-Dimethoxytrityl)-5-(6-biotinamidocaproamidomethyl)-1-(2-nitrophenyl)-ethyl]-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite(PC Biotin Phosphoramidite, from Glen Research Corp., www.glenres.com)was coupled to the 5′ terminus of a 21-mer oligoribonucleotide usingstandard phosphoramidite chemistry. Following the coupling step,oxidation, and cleavage from the resin, the caged oligoribonucleotidewas purified using RNase-free HPLC purification and verified using gelelectrophoresis analysis and mass spectrometry. An oligoribonucleotidecorresponding to the sense strand was also synthesized (5′CAUCAUCCCUGCCUCUACUdTdT 3′, SEQ ID NO:3), and equimolar amounts of thesense and caged antisense strands were annealed to form the cagedRNAi 1. An RNAi 1 siRNA which did not contain the caging group was alsosynthesized.

HeLa cells were lipofected with 100 nM RNAi 1, caged RNAi 1, or cagedRNAi 1 that had been uncaged in vitro, using Lipofectamine™ 2000(Invitrogen, www.invitrogen.com) according to the manufacturer'sinstructions. In brief, 5000 HeLa cells were plated evenly into eachwell of 96 well Coming Costar black clear bottom plates in 200 μL ofDulbecco modified Eagle medium (DMEM). The cells were incubated at 37°C. for 16-24 h, and then visually examined to ensure that each well was70-90% confluent and that the culture was evenly distributed in eachwell. For each well, 0.25 μg of the appropriate siRNA was diluted in 25μL OptiMEM and incubated at room temperature for 4 min; 0.5 μL ofLipofectamine 2000 was also diluted in 25 μL OptiMEM and incubated atroom temperature for 4 min. The siRNA and the lipofection reagent werethen combined, mixed gently, and incubated at room temperature for 20min, the volume was adjusted to 175 μL with OptiMEM, medium wasaspirated from the well containing the HeLa cells, and thesiRNA-lipofection reagent complex was added to the cells. Plates werethen incubated for 4 h at 37° C. with gentle shaking, then the mediumwas replaced with 200 μL fresh complete DMEM.

To cleave the caging group from the caged siRNA, following the 4 hlipofection with caged RNAi 1, cells were exposed from the bottom of thewell to 1 J/cm² 365 nm UV light. Uncaging light was produced by aBlueWave™ UV Spot Light System fitted with a Lightguide mount assembly,Cool Blue™ filter, and Lightguide rod lens assembly (Dymax Corp.,www.dymax.com, part numbers 38600, 38670, and 38699). Cells wereincubated at 37° C. and lysed at different time points after uncaging: 0h (immediately after uncaging), 6 h and 10 h after uncaging.

As controls, cells lipofected with unmodified RNAi 1, cells lipofectedwith caged RNAi 1 but not exposed to uncaging light, and cellslipofected with caged RNAi 1 that had previously been uncaged in vitroby exposure to 12 J/cm² of UV light were also maintained at 37° C. andlysed at 0 h (immediately following lipofection, corresponding to the 0h time point for the in vivo uncaged caged siRNA above), 6 h, and 10 h.

GAPDH mRNA was measured with a branched DNA assay using a QuantigeneExplore bDNA assay kit (Genospectra, Inc.) according to the instructionssupplied with the kit. To normalize for cell number, GAPDH expressionwas normalized to cyclophilin expression (also measured with a bDNAassay).

FIG. 29 Panel A shows GAPDH expression normalized to cyclophilinexpression in untransfected cells and cells transfected with: RNAi 1(unmodified), in vitro uncaged caged RNAi 1, caged RNAi 1 (transectedcells were not exposed to light), and in vivo uncaged caged RNAi 1, asmeasured by the bDNA assay at the indicated time points after uncaging(or just lipofection). FIG. 29 Panel B shows the relative GAPDH mRNAlevel as measured by the bDNA assay at the indicated time points afteruncaging (or just lipofection) of: RNAi 1 (unmodified), in vitro uncagedcaged RNAi 1, caged RNAi 1 (transected cells were not exposed to light),and in vivo uncaged caged RNAi 1. Expression is normalized to that ofcells transfected with the unmodified RNAi 1. Comparing relative GAPDHexpression in cells transfected with unmodified RNAi 1 and caged RNAi 1indicates that the caging group inhibits initiation of RNAi by the cagedsiRNA; levels of GAPDH mRNA are higher for cells transfected with thecaged siRNA but not exposed to light than for cells transfected withunmodified RNAi 1 at all three time points. Removal of the caging grouprestores the ability of the siRNA to participate in RNAi, since relativeGAPDH levels in cells transfected with caged RNAi 1 and then exposed toUV light are close to the levels in cells transfected with RNAi 1 at 6and 10 h after uncaging.

Enhanced Delivery and in vivo Photoactivation

RNAi 1 caged at the 5′ phosphate of the antisense strand (FIG. 28) wasproduced as described above. HeLa cells were lipofected with 3 nM cagedRNAi 1, caged RNAi 1 that had been uncaged in vitro, or a scrambledGAPDH negative control siRNA (Ambion catalog no. 4605, www.ambion.com),using Lipofectamine™ 2000 (Invitrogen, www.invitrogen.com). In brief,5000 HeLa cells were plated overnight in each well of 96 well blackclear bottom plates. Cells were transfected using Lipofectamine™ 2000, 3nM of the relevant siRNA, and 27 nM supercoiled pcDNA™3.1 plasmid(Invitrogen). Cells were exposed to the lipofection complex for 4 h inminimal media.

Mixing the siRNA with plasmid to form the lipofection complex permitsuse of lower concentrations of the siRNA than does forming thelipofection complex with siRNA in the absence of plasmid. Use of suchlower concentrations of caged siRNA can be advantageous, since anyuncaged siRNA contaminating the caged siRNA is thus also introduced intothe cells at a lower concentration, for example. As another example,using lower concentrations of the siRNA can decrease the risk ofoff-target effects (in which the siRNA affects expression of an mRNAthat is not the desired target).

To cleave the caging group from the caged siRNA, following the 4 hlipofection with caged RNAi 1, cells were exposed from the bottom of thewell to 1.4 J/cm² 365 nm UV light. Cells were incubated at 37° C. andlysed at different time points after uncaging: 0 h (immediately afteruncaging), 20 h and 44 h after uncaging.

As controls, cells lipofected with the scrambled negative control siRNA,cells lipofected with caged RNAi 1 but not exposed to uncaging light,and cells lipofected with caged RNAi 1 that had previously been uncagedin vitro by exposure to UV light were also maintained at 37° C. andlysed at 0 h (immediately following lipofection, corresponding to the 0h time point for the in vivo uncaged caged siRNA above), 20 h, and 44 h.

GAPDH mRNA was measured with a branched DNA assay using a QuantigeneExplore bDNA assay kit (Genospectra, Inc.) according to the instructionssupplied with the kit and normalized to cyclophilin expression.

FIG. 33 shows GAPDH expression normalized to cyclophilin expression incells transfected with: the scrambled GAPDH negative control siRNA,caged RNAi 1 (transected cells were not exposed to light), in vitrouncaged caged RNAi 1, and in vivo uncaged caged RNAi 1, as measured bythe bDNA assay at the indicated time points after uncaging (or justlipofection). Normalized expression is shown relative to GAPDHexpression in cells transfected with the scrambled GAPDH negativecontrol siRNA. FIG. 33 demonstrates that the photoactivated RNAi 1remains active in the cells for at least 44 h, while the unexposed cagedRNAi 1 remains inactive for at least 44 h. It also shows thattransfection with 3 nM of the caged RNAi in the presence of plasmid issufficient to reduce GAPDH expression following photoactivation.

In vivo Photoactivation at Different Light Dosages

RNAi 1 caged at the 5′ phosphate of the antisense strand was produced asdescribed above. HeLa cells were lipofected with 3 nM caged RNAi 1. Inbrief, 5000 HeLa cells were plated overnight in each well of 96 wellblack clear bottom plates. Cells were transfected using Lipofectamine2000, 3 nM caged RNAi 1, and 27 nM pcDNA™ 3.1 plasmid (Invitrogen).Cells were exposed to the lipofection complex for 4 h in minimal media.

Following the 4 h lipofection with caged RNAi 1, cells were exposed fromthe bottom of the well to varying energy densities of 365 nm UV light tocleave the caging group from the caged siRNA. Cells were incubated at37° C. and lysed at 0 h (immediately after uncaging) and 20 h afteruncaging. GAPDH mRNA was measured with a branched DNA assay using aQuantigene Explore bDNA assay kit (Genospectra, Inc.) according to theinstructions supplied with the kit and normalized to cyclophilinexpression.

FIG. 34 shows normalized GAPDH expression in cells transfected withcaged RNAi 1 as measured by the bDNA assay at 0 h and 20 h afteruncaging in vivo with varying doses of light (0.02 J/cm², 0.1 J/cm², 0.5J/cm², and 1.4 J/cm²). GAPDH expression in cells transfected with cagedRNAi 1 but not exposed to uncaging light (0.0 J/cm²) is also shown. FIG.34 demonstrates that suppression of GAPDH expression increases withhigher doses of light, indicating that increasing light dosagephotoactivates more of the caged siRNA. The caged siRNA can thus bepartially or completely photoactivated in vivo, as desired, bycontrolling the energy density of the uncaging light to which the cellsare exposed (e.g., by controlling the intensity of the uncaging lightand/or the duration of exposure).

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations.

All publications, patents, patent applications, and/or other documentscited in this application are incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent, patent application, and/or other document wereindividually indicated to be incorporated by reference for all purposes.

1. A composition comprising a caged RNA, the caged RNA comprising: anRNA capable of repressing translation of a target mRNA; and, one or morefirst caging groups associated with the RNA, the first caging groupsinhibiting the RNA from repressing translation of the target mRNA in acell comprising the caged RNA.
 2. The composition of claim 1, whereinthe RNA does not initiate degradation of the target mRNA in a cellcomprising the RNA.
 3. The composition of claim 1, wherein the RNA isdouble-stranded.
 4. The composition of claim 1, wherein the RNAcomprises at least an antisense strand, the antisense strand comprisinga first region which is complementary to a second region of the targetmRNA, the first region being interrupted by one or more nucleotideswhich are not complementary to the second region.
 5. The composition ofclaim 4, wherein the first region is interrupted by two, three, four, ormore nucleotides which are not complementary to the second region. 6.The composition of claim 4, wherein the second region is within the3′-untranslated region of the target mRNA.
 7. The composition of claim4, wherein the RNA comprises at least one double-stranded region, thedouble-stranded region comprising the antisense strand and a sensestrand.
 8. The composition of claim 7, wherein the sense strand iscompletely complementary to the antisense strand over thedouble-stranded region.
 9. The composition of claim 7, wherein the sensestrand is not completely complementary to the antisense strand over thedouble-stranded region.
 10. The composition of claim 7, wherein the RNAcomprises a first polyribonucleotide comprising the sense strand and asecond polyribonucleotide comprising the antisense strand.
 11. Thecomposition of claim 10, wherein the first polyribonucleotide comprisesbetween 17 and 29 nucleotides, the second polyribonucleotide comprisesbetween 17 and 29 nucleotides, and the double-stranded region comprisesbetween 17 and 29 base pairs.
 12. The composition of claim 11, whereinthe first polyribonucleotide comprises between 18 and 25 nucleotides,the second polyribonucleotide comprises between 18 and 25 nucleotides,and the double-stranded region comprises between 18 and 25 base pairs.13. The composition of claim 11, wherein the first polyribonucleotideand the second polyribonucleotide each comprise a two nucleotide TT 3′overhang.
 14. The composition of claim 10, wherein at least one of theone or more first caging groups is covalently attached to a 5′ hydroxylor a 5′ phosphate of the second polyribonucleotide.
 15. The compositionof claim 10, wherein the first caging group is covalently attached tothe first polyribonucleotide and to the second polyribonucleotide. 16.The composition of claim 15, wherein the first caging group is attachedto the 3′ end of the first polyribonucleotide and to the 5′ end of thesecond polyribonucleotide.
 17. The composition of claim 7, wherein theRNA comprises a self-complementary polyribonucleotide.
 18. Thecomposition of claim 1, comprising the target mRNA, a cell, a cellcomprising the target mRNA, or a cell comprising the caged RNA.
 19. Thecomposition of claim 1, wherein the first caging groups inhibit the RNAfrom repressing translation of the target mRNA by at least about 30%, atleast about 50%, at least about 75%, at least about 90%, at least about95%, or at least about 98%, as compared to the RNA in the absence of thefirst caging groups.
 20. The composition of claim 1, wherein the firstcaging groups prevent the RNA from repressing translation of the targetmRNA.
 21. The composition of claim 1, wherein removal of or an inducedconformational change in the first caging groups permits the RNA torepress translation of the target mRNA.
 22. The composition of claim 1,wherein the one or more first caging groups associated with the RNA arecovalently attached to the RNA.
 23. The composition of claim 1, whereinthe one or more first caging groups are removable by sonication,photoactivatable, or photolabile.
 24. The composition of claim 1,wherein the one or more first caging groups each comprises a firstbinding moiety; the composition comprising a second binding moiety thatcan bind at least one of the first binding moieties.
 25. The compositionof claim 1, wherein the RNA comprises at least one label.
 26. Thecomposition of claim 1, wherein the RNA is associated with a cellulardelivery module that can mediate introduction of the RNA into a cell.27. The composition of claim 26, wherein the cellular delivery modulecomprises a polypeptide, a PEP-1 pep tide, an amphipathic peptide, anMPG^(ΔNLS) peptide, a cationic peptide, a homopolymer of D-arginine, ahomopolymer of histidine, a homopolymer of lysine, a proteintransduction domain, a protein transduction domain derived from an HIV-1Tat protein, from a herpes simplex virus VP22 protein, or from aDrosophila antennapedia protein, a model protein transduction domain, ora model protein transduction domain comprising a homopolymer ofD-arginine.
 28. The composition of claim 26, wherein the cellulardelivery module is covalently attached to the RNA.
 29. The compositionof claim 28, wherein the cellular delivery module is attached to the RNAthrough a disulfide bond, or wherein the covalent attachment isreversible by exposure to light of a preselected wavelength.
 30. Thecomposition of claim 28, wherein the cellular delivery module comprisesa lipid or one or more myristoyl groups.
 31. The composition of claim26, wherein the cellular delivery module is associated with one or moresecond caging groups which inhibit the cellular delivery module frommediating introduction of the RNA into a cell.
 32. The composition ofclaim 1, wherein the RNA comprises a first polyribonucleotide comprisinga sense strand and a second polyribonucleotide comprising an antisensestrand, and wherein a cellular delivery module is covalently attached tothe second polyribonucleotide.
 33. The composition of claim 1, whereinthe first caging group is a cellular delivery module.
 34. Thecomposition of claim 1, wherein the caged RNA is bound to a matrix. 35.The composition of claim 34, wherein the matrix is a surface, and theRNA is bound to the surface at a predetermined location within an arraycomprising other RNAs.
 36. A kit for making the caged RNA of claim 1,comprising an RNA, one or more first caging groups, and instructions forassembling the RNA and the first caging groups to form the caged RNA,packaged in one or more containers; or comprising one or more firstcaging groups and instructions for assembling the first caging groupsand an RNA supplied by a user of the kit to form the caged RNA, packagedin one or more containers.
 37. A method of selectively attenuatingexpression of a target gene in a cell, the method comprising:introducing a caged RNA into the cell, the caged RNA comprising (a) anRNA capable of repressing translation of a target mRNA transcribed fromthe target gene, and (b) one or more first caging groups associated withthe RNA, the first caging groups inhibiting the RNA from repressingtranslation of the target mRNA in the cell; and, initiating repressionof translation of the target mRNA by exposing the cell to uncagingenergy, whereby exposure to the uncaging energy frees the RNA frominhibition by the caging groups.
 38. The method of claim 37, wherein theamount of the target mRNA present in the cell is not affected by thepresence of the RNA in the cell.
 39. The method of claim 37, whereinexposing the cell to uncaging energy comprises exposing the cell tolight of a first wavelength.
 40. The method of claim 39, whereinexposing the cell to light of the first wavelength comprises exposingthe cell to light wherein intensity of the light and duration ofexposure of the cell to the light are controlled such that a firstportion of the caged RNA is uncaged and a second portion of the cagedRNA remains caged.
 41. The method of claim 40, comprising exposing thecell to light of the first wavelength again.
 42. The method of claim 40,wherein the first portion is a selected amount.
 43. The method of claim37, comprising contacting the cell and a test compound, and wherein thecell is exposed to the uncaging energy at a preselected time point withrespect to a time at which the cell and the test compound are contacted.44. The method of claim 37, wherein the uncaging energy is directed at apreselected subset of a cell population comprising the cell.
 45. Themethod of claim 37, wherein the caged RNA comprises a cellular deliverymodule that can mediate introduction of the caged RNA into the cell, thecellular delivery module being associated with the RNA, and whereinintroducing the caged RNA into the cell comprises contacting the cellwith the caged RNA associated with the cellular delivery module.
 46. Themethod of claim 37, wherein the RNA comprises at least one label, themethod comprising detecting a signal from the label.
 47. A compositioncomprising a caged RNA, the caged RNA comprising: an RNA capable ofsilencing transcription of a target gene; and, one or more first caginggroups associated with the RNA, the first caging groups inhibiting theRNA from silencing transcription of the target gene in a cell comprisingthe caged RNA.
 48. A method of selectively attenuating expression of atarget gene in a cell, the method comprising: introducing a caged RNAinto the cell, the caged RNA comprising (a) an RNA capable of silencingtranscription of the target gene, and (b) one or more first caginggroups associated with the RNA, the first caging groups inhibiting theRNA from silencing transcription of the target gene in the cell; and,initiating silencing of transcription of the target gene by exposing thecell to uncaging energy, whereby exposure to the uncaging energy freesthe RNA from inhibition by the caging groups.
 49. A method ofselectively attenuating expression of a target gene in a cell, themethod comprising: introducing a first caged DNA and a second caged DNAinto the cell, the first caged DNA comprising a first DNA encoding anRNA sense strand and one or more caging groups associated with the firstDNA, the second caged DNA comprising a second DNA encoding an RNAantisense strand and one or more caging groups associated with thesecond DNA, the caging groups inhibiting transcription of the first andsecond DNAs, the first and second DNAs each comprising at least aportion of the target gene, and the sense and antisense strands being atleast partially complementary and able to form a duplex over at least aportion of their lengths; and, initiating translational repression bygenerating double-stranded RNA by exposing the cell to uncaging energy,whereby exposure to the uncaging energy frees the first and second DNAsfrom inhibition by the caging groups and permits transcription of thefirst and second DNAs to occur.
 50. The method of claim 49, wherein thesense strand comprises a first polyribonucleotide and the antisensestrand comprises a second polyribonucleotide, or wherein the sense andantisense strands comprise a single, self-complementarypolyribonucleotide.
 51. The method of claim 49, wherein exposing thecell to uncaging energy comprises exposing the cell to light of a firstwavelength.
 52. A composition, comprising: a protein transduction domaincovalently attached to an RNA; and, the RNA, which RNA comprises: (a) atleast one double-stranded region, the double-stranded region comprisinga sense strand and an antisense strand, the antisense strand comprisinga region which is substantially complementary to a region of a targetmRNA, or (b) a single polyribonucleotide strand comprising an antisensestrand, the antisense strand comprising a region which is substantiallycomplementary to a region of a target mRNA corresponding to the targetgene.
 53. The composition of claim 52, wherein the region of theantisense strand is completely complementary to the region of the targetmRNA.
 54. The composition of claim 52, wherein the region of theantisense strand which is substantially complementary to the region ofthe target mRNA comprises at least a first and a second subregion, eachof which is completely complementary to the target mRNA, flanking one ormore nucleotides which are not complementary to the target mRNA.
 55. Thecomposition of claim 54, wherein the first and second subregions flanktwo, three, four, or more nucleotides which are not complementary to thetarget mRNA.
 56. The composition of claim 52, comprising one or morefirst caging groups associated with the RNA, the first caging groupsinhibiting the RNA from repressing translation of the target mRNA in acell.
 57. A method of introducing an RNA into a cell, the methodcomprising: (a) providing a composition comprising (i) an RNA comprisingat least one double-stranded region, the double-stranded regioncomprising a sense strand and an antisense strand, the antisense strandcomprising a region which is substantially complementary to a region ofa target mRNA; or an RNA comprising a single polyribonucleotide strandcomprising an antisense strand, the antisense strand comprising a regionwhich is substantially complementary to a region of a target mRNA, and(ii) a protein transduction domain covalently attached to the RNA; and,(b) contacting the composition and the cell, whereby the proteintransduction domain mediates introduction of the RNA into the cell. 58.The method of claim 57, wherein the composition comprises one or morefirst caging groups associated with the RNA, the first caging groupsinhibiting the RNA from repressing translation of the target mRNA in thecell; the method comprising initiating translational repression of thetarget mRNA by exposing the cell to uncaging energy of a first type,whereby exposure to the uncaging energy frees the RNA from inhibition bythe first caging groups.